Methods for sem inspection of fluid containing samples

ABSTRACT

A method of visualizing a sample in a wet environment including introducing a sample into a specimen enclosure in a wet environment and scanning the sample in the specimen enclosure in a scanning electron microscope, thereby visualizing the sample.

REFERENCE TO CO-PENDING APPLICATIONS

Applicant hereby claims priority of U.S. Provisional Patent ApplicationSer. No. 60/393,747, filed on Jul. 8, 2002, entitled “Quantative PatternAnalysis of Molecules on Intact Cells Using Automated SEM”, U.S.Provisional Patent Application Serial No. 60/448,808, filed on February20, 2003, entitled “A Specimen Enclosure for a Scanning ElectronMicroscope”, Israel Patent Application Serial No. 150054, filed on Jun.5, 2002, entitled “Device for Fluorescent Imaging of Biological SamplesUsing a Scanning Electron Microscope and Fluorescent or ScintillationMarkers” and Israel Patent Application Serial No. 150055, filed on Jun.5, 2002, entitled “Automation Compatible Devices for Scanning ElectronMicroscopy Imaging of Samples in a Wet Environment”.

FIELD OF THE INVENTION

The present invention relates to SEM inspection of fluid containingsamples generally and more particularly to methods of visualizingsamples in a wet environment.

BACKGROUND OF THE INVENTION

The following documents are believed to represent the current state ofthe art, and the disclosures of each are incorporated by reference:

-   1. Tucker, J. A. (2000) The continuing value of electron microscopy    in surgical pathology. Ultrastructural Pathology 24:383-9-   2. Mathews, R. A. and Donald, A. M. (2002) Conditions for imaging    emulsions in the environmental scanning electron microscope.    Scanning 24:75-85.-   3. Mittler, M. A., Walters, B. C. and Stopa, E. G. (1996) Observer    reliability in histological grading of astrocytoma stereotactic    biopsies. J. Neuroswg 85:1091-4.-   4. Levit-Binnun, N., Lindner A. B., Zik O., Eshhar Z. and    Moses, E. (2003) Quantitative detection of protein arrays. Anal    Chem. 75:1436-41.-   5. Becker, R. P.. and Sogard, M. (1979) Visualization of subsurface    structures in cells and tissues by backscattered electron imaging.    Scan. Electron. Microsc. 1979 (II): 835-70.-   6. Sedar, A. W., Silver, M. J. and Ingerman-Wolenski, C. M. (1983)    Backscattered electron imaging to visualize arterial endothelial    detachment in the scanning electron microscope. Scan. Electron.    Microsc. 1983 (II): 969-74.-   7. Burns, W. A., Zimmerman, H. J., Hammond, J., Howatson, A. Katz, A    and White, J. (1975) The clinician's view of diagnostic electron    microscopy. Hum. Pathol. 6:467-78.-   8. Gyorkey, F., Min, K. W., Krisko, I. And Gyorkey, P. (1975) The    usefulness of electron microscopy in the diagnosis of human tumors.    Hum. Pathol. 6:421-41.-   9. Hayat, M. A. (2000) Principles and Techniques of Electron    Microscopy-Biological Applications (Fourth edition; Cambridge    University Press)-   10. Hermann, R., Walther, P. and Müller, M. (1996)    Immunogold-labeling in SEM. Histochem. Cell. Biol. 106:31-39.-   11. Spargo, B. H. (1975) Practical use of electron microscopy for    the diagnosis of glomerular disease. Hum. Pathol. 6:405-20.-   12. Gu, X. and Heirera, G. A. (2002) The value of electron    microscopy in the diagnosis of IgA nephropathy. Ultrastruct Pathol.    26:203-10-   13. Fisher, C, Ramsay, A D, Griffiths, M and McDougall, J. (1985) An    assessment of the value of electron microscopy in tumor    diagnosis. J. Clin Pathol. 38:403-8-   14. Brocker, W, Pfefferkorn G. (1975) Applications of the    cathodoluminescence method in biology and medicine. Scan. Electron.    Microsc. 1979;(II): 125-32-   15. Carlen, B. and Englund, E. (2001) Diagnostic value of electron    microscopy in a case of juvenile neuronal ceroid lipofuscinosis.    Ultrastruct. Pathol. 25:285-8-   16. Hollinshead M, Sanderson J. & Vaux D. J. (1997). Anti-biotin    antibodies offer superior organelle-specific labeling of    mitochondria over avidin or streptavidin. J. Histochem. Cytochem.    45:1053-7-   17. Kristiansen, E. and Madsen, C. (1995) Induction of protein    droplet (alpha-2 microglobulin) nephropathy in male rats after    short-term dosage with 1,8-cineole and 1-limonene. Toxicol. Letters    80:147-52.-   18. Goldstein, J. I., Newbury, D. E., Echlin, P., and Joy, D.    (1992). Scanning Electron Microscopy and X-ray microanalysis: a text    for biologists, matrials scientists, and geologists. Plenum Press,    1992.-   19. Schlessinger, J. (2002) Ligand-induced, receptor-mediated    dimerization and activation of EGF receptor. Cell 110:669-72.

U.S. patents documents:

U.S. Pat. Nos. 3,218,459; 3,378,684; 4,037,109; 4,071,766; 4,115,689;4,448,311; 4,587,666; 4,596,928; 4,618,938; 4,705,949; 4,720,622;4,720,633; 4,880,976; 4,929,041; 4,992,662; 5,103,102; 5,250,808;5,323,441; 5,326,971; 5,362,964; 5,406,087; 5,412,211; 5,811,803;5,898,261; 5,945,672; 6,025,592; 6,072,178; 6,114,695; 6,130,434;6,365,898 and 6,452,177.

Published PCT application WO02/14830-PCT/IL01/00764.

Microscopic examination of biological cells and tissues is a centraltool in clinical diagnosis as well as in diverse areas of research inthe life sciences. Light microscopy (LM) is performed with thin (severalmicron) samples, which may include cells, acellular material, or thinlayers or sections of tissue, which may be stained with contrast agentssuch as chemicals or antibodies. Transmission electron microscopy (TEM)usually requires specially prepared ultrathin sections (0.1 micron orless), and reveals a wealth of subcellular information. Each of theaforementioned techniques has limitations: the resolution of lightmicroscopy is limited by diffraction to approximately 0.25 microns andthe use of TEM is encumbered by extensive processing of the sample,which may alter its structure significantly. Preparation of samples forstandard TEM also requires specific skills and takes at least a few daysto achieve. The very thin slices present a very limited, and oftenarbitrary, portion of the sample, necessitating the imaging of multipleserial sections.

High resolution images can also be achieved by scanning electionmicroscopy (SEM), in which a focused electron beam scans the samplesequentially, and ensuing signals are used to generate an image. Mostoften, secondary electrons are detected, yielding information on thesurface topography of the sample. Detection of backscattered electronsyields information on material distribution of a region of the samplelying a short distance below the surface, typically up to a few microns.SEM is a reflective mode of imaging, in the sense that electrons do notneed to traverse the sample to yield an image. Therefore, samples can beof any thickness, and do not need to be sectioned. However, samples mustbe placed in a vacuum environment to allow unimpeded motion of thescanning electron beam; therefore, the sample has to be extensivelydehydrated and dried. Furthermore, when dried, biological and otherorganic samples become electrically insulating, leading to artifacts dueto charging of the samples by the electron beam. Consequently, samplesare usually coated with a conductive layer of carbon or metal.

One approach to observing biological or other wet samples withoutextensive drying has been the development of environmental SEM andsimilar techniques. These methods are based on differential pumping andmultiple apertures, allowing a localized pressure close to the vaporpressure of water in the close vicinity of the sample, while maintaininga high vacuum through most of the path of the scanning electron beam. Inthese methods the sample is exposed to a partial vacuum, and maintenanceof the hydrated state requires both a low temperature and complicated,manual maintenance of the right pressure. Indeed, the paucity ofpublished reports of biological research using this technique attests tothe difficulty in obtaining consistent results of a high standard

High resolution imaging has wide-spread applications in biology,including imaging of cells, tissues, microbes, and viruses, as well as acellular samples such as biological, environmental or industrial fluids,emulsions and suspensions.

A recent review discusses the use of electron microscopy in clinicaldiagnosis (Tucker J. A., 2000). It is found that in a small butsignificant proportion of cases (3-8%) proper diagnosis can only be madebased on electron microscopy, this is particularly pronounced inoncology and in selected areas such as kidney diseases (Tucker 2000).These numbers are probably an underestimate, since the use of electronmicroscopy is not primarily limited by lack of utility, but byconsiderations of cost, the time needed to produce results, and the lowthroughput. Thus, there is a significant need for an imaging system forbiological tissues and cells that achieves electron microscopicresolution with sample preparation procedures comparable with those oflight microscopy.

SUMMARY OF THE INVENTION

Co-pending patent application PCT/IL01/01108, by one of the presentinventors, entitled “device and method for the examination of samples ina non-vacuum environment using a scanning electron microscope”,discloses a non-vacuum Scanning Electron Microscope (SEM) device thatenables the imaging of wet samples in a wet environment, atnear-atmospheric pressure and a wide range of temperatures. Thisobviates the need for extensive sample preparation procedures, thuscombining some advantages of electron microscopy, such as highresolution and high contrast or spatial signal to noise ratio, withadvantages of light microscopy, such as ease and speed of samplepreparation. This is accomplished by the use of a sample containercovered by a thin partition membrane that is permeable to electrons butis fluid impermeable and sufficiently strong to withstand the pressuredifference between the interior of the container, which is typically oneatmosphere, and the vacuum in the imaging region of an SEM. This type ofmembrane is referred to hereinbelow as a partition membrane or as anelectron-permeable, fluid impermeable, membrane.

The aforementioned features of non-vacuum SEM are applied to theobservation of fluid-containing samples, especially biological samples,in the following manner:

1. Separation of the sample from the vacuum allows direct visualizationof wet samples. This immediately obviates the need for all dehydrationprocedures, including water replacement and critical-point drying. Thewet state most closely resembles the native state of the sample,preserving features that are distorted or destroyed during dehydration.This advantage is particularly important in the observation of tissues,where the true architecture involves both cells and extracellularmatrix. In addition, the presence of fluid in and around the sampleallows efficient dissipation of electrical charge and of excess heat.This eliminates artifacts due to sample charging, as well as thermaldamage.

2. Electron microscopy of biological tissues is most frequently done intwo imaging modes. Transmission electron microscopy (TEM) utilizeselectrons transmitted through the sample; the entire thickness of thesample contributes to the image. Transmission techniques impose a severeconstraint on the thickness of the samples: typically, 50 nm, which canbe increased to 3 μm in ultrahigh voltage microscopes. Scanning electronmicroscopy uses a reflective mode, most frequently detecting secondaryelectrons that image only the surface topography of the sample. Thenon-vacuum SEM technique uses backscattered electron detection in ascanning electron microscope. The electron beam penetrates into thesample, and the backscattered electrons reveal sample features beyondthe sample surface to a depth of up to a few microns. Thus, although anelectron scanning/reflecting mode of imaging is employed, the image isnot limited to the surface, and internal structure of the sample isrevealed Furthermore, because detection is done in a reflective mode,any material lying beyond the interaction volume has no effect onimaging. Therefore, the samples can be of a thickness far exceeding theimaged region. Typically, a tissue fragment several millimeters thickcan be viewed; only the material layer of a few micrometers or less thatis closest to the surface contributes to the scanned image, withoutinterference from the bulk of the sample. The thickness of the imagedregion can be modulated by varying the acceleration voltage of theelectron beam. Non-vacuum SEM thus yields “virtual sections” without theneed for actual sectioning of the sample. This eliminates the need forembedding or freezing the sample, which are otherwise required to enablesectioning of the sample. Finally, the dependence of electronbackscattering efficiency on the material composition of the sample(through the atomic number Z) creates contrast even in the absence ofheavy metal staining that is characteristic of TEM imaging. Subcellularorganelles can be distinguished based on differences in localconcentrations of lipids, phosphates, and salts within biologicalsamples; and a wide variety of stains and labels can be used to enhancecontrast.

An additional capability of the non-vacuum SEM technology is concurrentdetection of light emitted from the sample while scanning with theelectron beam. The scanning electron beam excites molecules in thesample, which may then emit light at characteristic wavelengths(cathodoluminescence). The light intensity is then used to derive animage of the distribution of scintillating molecules, either endogenousto the biological sample or labels that can be introduced extraneously.This image is obtained simultaneously with the imaging by backscatteredelectrons (BSE), at a resolution limited by electron-matter interactionsand not by light diffraction. Similarly, X-rays emitted from the scannedsample can be detected using conventional detectors and methods,yielding additional information on the material composition anddistribution of the fully hydrated sample. Specifically, X-ray analysiscan be used to localize and quantitate regions containing metals such ascalcium, iron, sodium, potassium, copper or zinc, or other elements suchas iodine, sulfur, or phosphor.

It is another objective of the present invention to provide means forautomated electron microscopy of wet samples, and specifically ofbiological samples. Such automated microscopy has been widely applied inthe semiconductor industry. The main barrier to the application ofautomated electron microscopy to wet samples is the need to employsample preparation procedures such as drying, embedding, sectioning orcoating, which are highly complex and not amenable to automation. Thepresent invention provides means for direct imaging of wet samples in ascanning electron microscope, thus obviating the need for theaforementioned preparative procedures. The present invention thusprovides means for automated electron microscopy of wet samples.

There is thus provided in accordance with a preferred embodiment of thepresent invention a method of visualizing a sample in a wet environmentincluding introducing a sample into a specimen enclosure in a wetenvironment and scanning the sample in the specimen enclosure in ascanning electron microscope, thereby visualizing the sample.

There is also provided in accordance with another preferred embodimentof the present invention a method of visualization of a sample in a wetenvironment including introducing a sample into a specimen enclosure ina wet environment and scanning the sample in the specimen enclosure in ascanning electron microscope at multiple different electron energylevels.

There is further provided in accordance with yet another preferredembodiment of the present invention a method of visualizing at least onecomponent in a multi-component sample in a wet environment includingintroducing a multi-component sample into a specimen enclosure in a wetenvironment and scanning the multi-component sample in the specimenenclosure in a scanning electron microscope, thereby visualizing atleast one component of the multi-component sample.

There is even further provided in accordance with still anotherpreferred embodiment of the present invention a method for visualizationof a sample in a wet environment including obtaining a wet sample,scanning the wet sample in a scanning electron microscope at aresolution which is not limited by the diffraction limit of light anddetecting light emitted from the wet sample.

In accordance with another preferred embodiment of the present inventionthe method also includes enhancing contrast between plural elements inthe sample prior to scanning of the sample. Preferably, the enhancingincludes specific labeling of at least some of the plural elements inthe sample. Alternatively, the enhancing includes specific labeling ofmolecules in the sample. In accordance with another preferred embodimentthe enhancing includes specific labeling of receptors in the sample.Alternatively, the enhancing includes specific labeling of organelles inthe sample. Additionally, the enhancing includes specific labeling ofbinding sites in the sample. Preferably, the enhancing includes specificlabeling of structural elements in the sample. Alternatively, theenhancing includes specific labeling of functional elements in thesample.

In accordance with yet another preferred embodiment of the presentinvention the scanning visualizes elements in the sample having contrastdue to differences in atomic numbers of constituent atoms thereof.

In accordance with still another preferred embodiment of the presentinvention, the enhancing introduces differences in atomic numbers ofconstituent atoms of elements of the samples.

Preferably, the scanning visualizes lipid containing entities in thesample. Alternatively, the scanning visualizes nucleic acid containingentities in the sample. Additionally, the scanning visualizes proteincontaining entities in the sample. In accordance with another preferredembodiment of the present invention the scanning visualizes carbohydratecontaining entities in the sample. Alternatively, the scanningvisualizes metal containing entities in the sample. Additionally, thescanning visualizes iodine containing entities in the sample.

In accordance with another preferred embodiment of the present inventionthe sample is a biological sample. Additionally, the biological sampleincludes cells in a liquid. Preferably, the sample includes lipidswithin cells and the scanning visualizes the lipids.

In accordance with yet another preferred embodiment of the presentinvention the scanning visualizes cells in the sample. Alternatively,the scanning visualizes tissue in tissue slices. Preferably, thescanning visualizes tissue. In accordance with another preferredembodiment of the present invention the scanning visualizes cells in thesample which are adherent to an electron beam permeable membrane.

In accordance with another preferred embodiment of the present inventionthe specimen enclosure includes an electron beam permeable membrane andthe method also includes growing cells on the electron beam permeablemembrane prior to the scanning. Alternatively or additionally, thespecimen enclosure includes an electron beam permeable membrane and themethod also includes manipulating cells on the electron beam permeablemembrane prior to the scanning. Additionally or alternatively, thescanning visualizes an outcome of introduction of an extrinsic moleculeto the sample

In accordance with another preferred embodiment of the present inventionthe method also includes detecting electromagnetic radiation emittedfrom the sample as a result of the scanning. Additionally, the methodalso includes analyzing the electromagnetic radiation. Preferably, theanalyzing the electromagnetic radiation includes spectral analysis.

In accordance with another preferred embodiment of the present inventionthe method also includes detecting an electron beam backscattered fromthe sample as well as electromagnetic radiation emitted from the sampleas a result of the scanning. Preferably, the electromagnetic radiationincludes X-ray radiation. Alternatively or additionally, theelectromagnetic radiation includes visible radiation. Additionally oralternatively, the electromagnetic radiation includes radiation having awavelength within the range of 200-1000 nm. Alternatively oradditionally, the electromagnetic radiation includes radiation providinginformation relating to molecular structure of the sample. Additionallyor alternatively, the electromagnetic radiation includes radiationproviding information relating to material distribution within thesample.

In accordance with another preferred embodiment of the present inventionthe scanning includes scanning the sample in the specimen enclosure in ascanning electron microscope at multiple different electron energylevels. Additionally, the method also includes reconstructing athree-dimensional image of the sample by using multiple visualizationsof the sample at the multiple different electron energy levels.

In accordance with another preferred embodiment of the present inventionthe sample is a multi-component sample and the scanning includesscanning the multi-component sample in the specimen enclosure in ascanning electron microscope, thereby visual zing at least one componentof the multi-component sample. Additionally or alternatively, the methodalso includes obtaining the sample to be inspected, the sample being ina wet environment.

There is still further provided in accordance with another preferredembodiment of the present invention a method for manufacture of abiocompatible implant including forming a biocompatible implant of abiocompatible material, inspecting at least a portion of thebiocompatible implant in a scanning electron microscope in a wetenvironment, analyzing results of the inspecting and classifying theinspected biocompatible implant in accordance with results of theanalyzing.

In accordance with another preferred embodiment of the presentinvention, the inspecting includes introducing a sample including atleast the portion into a specimen enclosure in a wet environment andscanning the sample in the specimen enclosure in the scanning electronmicroscope, thereby visualizing the sample.

There is also provided in accordance with yet another preferredembodiment of the present invention a method for detection,identification or characterization of microbiological entities includingobtaining a wet sample containing at least one microbiological entity,scanning the wet sample in a scanning electron microscope while in anenvironment characterized by a pressure exceeding the vapor pressure ofwater and analyzing results of the scanning.

Preferably, the scanning includes introducing the wet sample includingthe at least one microbiological entity into a specimen enclosure in awet environment and scanning the wet sample in the specimen enclosure inthe scanning electron microscope, thereby visualizing the wet sample.

In accordance with a preferred embodiment of the present invention thewet sample includes urine. Alternatively, the wet sample includes feces.Additionally, the wet sample includes blood. Additionally oralternatively, the wet sample includes sputum. In accordance withanother preferred embodiment of the present invention, the wet sampleincludes lavage of the respiratory system. Additionally, the wet sampleincludes a tissue biopsy. Alternatively, the wet sample includes anenvironmental sample. Additionally or alternatively, the wet sampleincludes cerebro-spinal fluid. In accordance with yet another preferredembodiment of the present invention, the wet sample includes a soilsample. Alternatively, the wet sample includes food. Additionally, thewet sample includes industrial products. Alternatively or additionally,the wet sample includes a medical, industrial or household device.

In accordance with another preferred embodiment of the present inventionthe method also includes specific staining of the sample. Additionally,the method also includes treating the microbiological entity withchemicals. Alternatively or additionally, he method also includesapplying radiation to the microbiological entity. Additionally oralternatively, the analyzing includes analyzing the morphology of themicrobiological entity.

There is further provided in accordance with yet another preferredembodiment of the present invention a method for characterization ofbiofilms including obtaining a wet sample of a biofilm, scanning the wetsample of a biofilm in a scanning electron microscope and analyzingresults of the scanning.

Preferably, the scanning includes introducing the wet sample includingthe biofilm into a specimen enclosure in a wet environment and scanningthe wet sample in the specimen enclosure in the scanning electronmicroscope, thereby visualizing the wet sample.

There is even further provided in accordance with still anotherpreferred embodiment of the present invention a method of visualizing asample including obtaining a sample and scanning the sample in a wetenvironment in a scanning electron microscope while in an environmentcharacterized by a pressure exceeding the vapor pressure of water,without an intermediate solidifying, coating or slicing step therebyvisualizing the sample.

There is yet further provided in accordance with another preferredembodiment of the present invention a method of visualizing a sampleincluding obtaining a sample and scanning the sample in a wetenvironment in a scanning electron microscope while in an environmentcharacterized by a pressure exceeding the vapor pressure of water,without morphologically impacting preparation prior to scanning.

There is still further provided in accordance with yet another preferredembodiment of the present invention a method of visualizing a sampleincluding obtaining a sample and scanning the sample in a wetenvironment in a scanning electron microscope while in an environmentcharacterized by a pressure exceeding the vapor pressure of water,following at most fixing and staining before scanning.

There is also provided in accordance with still another preferredembodiment of the present invention a method of visualizing a sampleincluding obtaining a sample and scanning the sample in a wetenvironment in a scanning electron microscope while in an environmentcharacterized by a pressure exceeding the vapor pressure of water,following at most treatment with at least one aqueous solution prior toscanning.

There is further provided in accordance with yet another preferredembodiment of the present invention a method of visualizing a sampleincluding obtaining a sample and scanning the sample in a wetenvironment in a scanning electron microscope while in an environmentcharacterized by a pressure exceeding the vapor pressure of water,without having been treated with a non-aqueous solution prior toscanning.

There is even further provided in accordance with another preferredembodiment of the present invention a method of visualizing a sampleincluding obtaining a sample of thickness exceeding 20 microns andscanning the sample in a scanning electron microscope while in anenvironment characterized by a pressure exceeding the vapor pressure ofwater.

There is also provided in accordance with yet another preferredembodiment of the present invention a method of inspecting adiposetissue including obtaining a sample of adipose tissue and scanning thesample of adipose tissue in a scanning electron microscope while in anenvironment characterized by a pressure exceeding the vapor pressure ofwater.

Preferably, the sample is not stained prior to the scanning.

There is further provided in accordance with still another preferredembodiment of the present invention a method of inspecting adiposetissue including obtaining a sample of adipose tissue and scanning thesample of adipose tissue in a scanning electron microscope without thesample having been stained prior to the scanning.

There is even further provided in accordance with another preferredembodiment of the present invention a method for visualization of asample including scanning the sample in a scanning electron microscopewhile in an environment characterized by a pressure exceeding the vaporpressure of water, the scanning taking place over an area of the samplewhich exceeds 25 square millimeters without displacement of the samplerelative to a stage of the scanning electron microscope.

There is also provided in accordance with yet another preferredembodiment of the present invention a method of inspecting anextracellular matrix including obtaining a sample of an extracellularmatrix and scanning the sample of the extracellular matrix in a scanningelectron microscope while in an environment characterized by a pressureexceeding the vapor pressure of water.

There is further provided in accordance with still another preferredembodiment of the present invention a method of inspecting epithelialtissue including obtaining a sample of epithelial tissue and scanningthe sample of the epithelial tissue in a scanning electron microscopewhile in an environment characterized by a pressure exceeding the vaporpressure of water.

There is even further provided in accordance with another preferredembodiment of the present invention a method of inspecting kidney tissueincluding obtaining a sample of kidney tissue and scanning the sample ofthe kidney tissue in a scanning electron microscope while in anenvironment characterized by a pressure exceeding the vapor pressure ofwater.

There is also provided in accordance with yet another preferredembodiment of the present invention a method of inspecting a tissuebiopsy including obtaining a sample of a tissue biopsy and scanning thesample of the tissue biopsy in a scanning electron microscope while inan environment characterized by a pressure exceeding the vapor pressureof water.

There is further provided in accordance with still another preferredembodiment of the present invention a method of inspecting biologicalmaterial including immunolabeling biological material and scanning theimmunolabeled biological material in a scanning electron microscopewhile in an environment characterized by a pressure exceeding the vaporpressure of water.

There is also provided in accordance with still another preferredembodiment of the present invention a method of inspecting tissueincluding immunolabeling tissue and scanning the immunolabeled tissue ina scanning electron microscope while in an environment characterized bya pressure exceeding the vapor pressure of water.

There is further provided in accordance with yet another preferredembodiment of the present invention a method of inspecting a sampleincluding scanning the sample in a scanning electron microscope while inan environment characterized by a pressure exceeding the vapor pressureof water and inspecting the sample using light microscopy.

There is also provided in accordance with another preferred embodimentof the present invention a method of analyzing toxic effects of exposureto a chemical or combination of chemicals including subjecting anexperimental animal to the exposure to the chemical or combination ofchemicals, obtaining a sample from the experimental animal and scanningthe sample from the experimental animal in a scanning electronmicroscope while in an environment characterized by a pressure exceedingthe vapor pressure of water.

There is further provided in accordance with yet another preferredembodiment of the present invention a method of analyzing toxic effectsfollowing exposure to environmental conditions including identify atleast one individual that was exposed to the environmental conditions,obtaining at least one sample from at least one of the at least oneindividual and scanning the at least one sample in a scanning electronmicroscope while in an environment characterized by a pressure exceedingthe vapor pressure of water.

There is even further provided in accordance with still anotherpreferred embodiment of the present invention a method of characterizingchemical entities including applying a chemical entity to cells in aSEM-compatible sample enclosure and scanning the cells in a scanningelectron microscope while in an environment characterized by a pressureexceeding the vapor pressure of water.

Preferably, the method also includes analyzing changes in cell shape.Additionally or alternatively, the method also includes analyzing thecytoskeleton of the cells. Additionally, the method also includesanalyzing the distribution of biomolecules in the cells.

In accordance with another preferred embodiment of the present inventionthe method also includes detecting x-rays from a region of the sampleand analyzing the x-rays to detect the presence of at least one ofiodine, metals and phosphorous in the sample. Preferably, the methodalso includes determining the concentration of the at least one ofiodine, metals, and phosphorus.

In accordance with yet another preferred embodiment of the presentinvention the sample is impinged upon by electrons passing through anelectron-permeable, fluid-impermeable membrane. Additionally, the methodalso includes urging the sample against the electron-permeable,fluid-impermeable membrane.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be understood and appreciated more fully fromthe following detailed description, taken in conjunction with thedrawings in which:

FIGS. 1A & 1B are oppositely facing simplified exploded view pictorialillustrations of a disassembled SEM compatible sample containerconstructed and operative in accordance with a preferred embodiment ofthe present invention;

FIGS. 2A & 2B are oppositely facing simplified partially pictorial,partially sectional illustrations of a subassembly of the container ofFIGS. 1A & 1B;

FIGS. 3A & 3B are oppositely facing simplified exploded view pictorialillustrations of the SEM compatible sample container of FIGS. 1A-2B in apartially assembled state;

FIGS. 4A & 4B are oppositely facing simplified pictorial illustrationsof the SEM compatible sample container of FIGS. 1A-3B in a fullyassembled state;

FIGS. 5A & 5B are oppositely facing simplified partially pictorial,partially sectional illustrations taken along lines VA-VA and VB-VB,respectively, in FIGS. 3A & 3B;

FIGS. 6A, 6B & 6C are three sectional illustrations showing theoperative orientation of the SEM compatible sample container of FIGS.1A-5B at three stages of operation;

FIGS. 7A, 7B, 7C, 7D and 7E are simplified sectional illustrations ofcell growth, liquid removal, liquid addition, sealing and insertion intoa SEM respectively using the SEM compatible sample container of FIGS.1A-6C;

FIGS. 8A, 8B and 8C are simplified sectional illustrations of liquidcontaining samples, sealing and insertion into a SEM respectively usingthe SEM compatible sample container of FIGS. 1A-6C;

FIG. 9 is a simplified pictorial and sectional illustration of a SEMinspection of a sample using the SEM compatible sample container ofFIGS. 1A-6C;

FIG. 10 is a greatly enlarged simplified schematic illustration of theSEM inspection of a sample in the context of FIG. 9;

FIGS. 11A & 11B are oppositely facing simplified exploded view pictorialillustrations of a disassembled SEM compatible sample containerconstructed and operative in accordance with another preferredembodiment of the present invention;

FIGS. 12A & 12B are oppositely facing simplified partially pictorial,partially sectional illustrations of a subassembly of the container ofFIGS. 11A & 11B;

FIGS. 13A & 13B are oppositely facing simplified exploded view pictorialillustrations of the SEM compatible sample container of FIGS. 11A-12B ina partially assembled state;

FIGS. 14A & 14B are oppositely facing simplified pictorial illustrationsof the SEM compatible sample container of FIGS. 11A-13B in a fullyassembled state;

FIGS. 15A & 15B are oppositely facing simplified partially pictorial,partially sectional illustrations taken along lines XVA-XVA and XVB-XVB,respectively, in FIGS. 13A & 13B;

FIGS. 16A, 16B & 16C are three sectional illustrations showing theoperative orientation of the SEM compatible sample container of FIGS.11A-15B at three stages of operation;

FIGS. 17A, 17B, 17C, 17D and 17E are simplified sectional illustrationsof cell growth, liquid removal, liquid addition, sealing and insertioninto a SEM respectively using the SEM compatible sample container ofFIGS. 11A-16C;

FIGS. 18A, 18B and 18C are simplified sectional illustrations of liquidcontaining samples, sealing and insertion into a SEM respectively usingthe SEM compatible sample container of FIGS. 11A-16C;

FIG. 19 is a simplified pictorial and sectional illustration of a SEMinspection of a sample using the SEM compatible sample container ofFIGS. 11A-16C;

FIG. 20 is a greatly enlarged simplified schematic illustration of theSEM inspection of a sample in the context of FIG. 19;

FIGS. 21A and 21B are simplified exploded view illustrations of apre-microscopy multi-sample holder in use with SEM compatible samplecontainers of the type shown in FIGS. 1A-20;

FIGS. 22A and 22B are simplified illustrations of the pre-microscopymulti-sample holder of FIGS. 21A & 21B respectively uncovered andcovered in an assembled state;

FIGS. 23A, 23B and 23C are simplified illustrations of thepre-microscopy multi-sample holder of FIGS. 21A-22B respectivelyassociated with a suction device and pipettes;

FIGS. 24A, 24B and 24C are simplified illustrations of a microscopymulti-sample holder in use with a SEM compatible sample dish of the typeshown in FIGS. 1A-10;

FIGS. 25A, 25B and 25C are simplified illustrations of a microscopymulti-sample holder in use with a SEM compatible sample dish of the typeshown in FIGS. 11A-20;

FIGS. 26A and 26B are simplified illustrations of a microscopymulti-sample holder defining a plurality of SEM compatible samplecontainers in accordance with a preferred embodiment of the presentinvention;

FIGS. 27A and 27B are simplified illustrations of a microscopymulti-sample holder defining a plurality of SEM compatible samplecontainers in accordance with a preferred embodiment of the presentinvention;

FIG. 28 is a simplified illustration of a SEM based sample inspectionsystem constructed and operative in accordance with a preferredembodiment of the present invention;

FIG. 29 is a simplified illustration of a SEM based sample inspectionsystem constructed and operative in accordance with another preferredembodiment of the present invention;

FIG. 30 is a simplified illustration of a SEM based sample inspectionsystem constructed and operative in accordance with yet anotherpreferred embodiment of the present invention;

FIGS. 31A & 31B are oppositely facing simplified exploded view pictorialillustrations of a disassembled SEM compatible sample containerconstructed and operative in accordance with another preferredembodiment of the present invention;

FIGS. 32A & 32B are oppositely facing simplified partially pictorial,partially sectional illustrations of a subassembly of the container ofFIGS. 31A & 31B;

FIGS. 33A & 33B are oppositely facing simplified exploded view pictorialillustrations of the SEM compatible sample container of FIGS. 31A-32B ina partially assembled state;

FIGS. 34A & 34B are oppositely facing simplified pictorial illustrationsof the SEM compatible sample container of FIGS. 31A-33B in a fullyassembled state;

FIGS. 35A & 35B are oppositely facing simplified partially pictorial,partially sectional illustrations taken along lines XXXVA-XXXVA andXXXVB-XXXVB, respectively, in FIGS. 33A & 33B;

FIGS. 36A, 36B & 36C are three sectional illustrations showing theoperative orientation of the SEM compatible sample container of FIGS.31A-35B at three stages of operation;

FIG. 37 is a simplified sectional and pictorial illustration of tissuecontaining samples and insertion into a SEM using the SEM compatiblesample container of FIGS. 31A-36C;

FIGS. 38A, 38B, 38C and 38D are simplified sectional illustrationsshowing the operative orientation of a SEM compatible sample containerat various stages of operation and insertion into a SEM using the SEMcompatible sample container constructed and operative in accordance withanother preferred embodiment of the present invention;

FIG. 39 is a simplified pictorial and sectional illustration of a SEMinspection of a sample using the SEM compatible sample container ofFIGS. 31A-37;

FIG. 40 is a greatly enlarged simplified schematic illustration of theSEM inspection of a sample in the context of FIG. 39;

FIGS. 41A & 41B are oppositely facing simplified exploded view pictorialillustrations of a disassembled SEM compatible sample containerconstructed and operative in accordance with another preferredembodiment of the present invention;

FIGS. 42A & 42B are oppositely facing simplified partially pictorial,partially sectional illustrations of a subassembly of the container ofFIGS. 41A & 41B;

FIGS. 43A & 43B are oppositely facing simplified exploded view pictorialillustrations of the SEM compatible sample container of FIGS. 41A-42B ina partially assembled state;

FIGS. 44A & 44B are oppositely facing simplified pictorial illustrationsof the SEM compatible sample container of FIGS. 41A-43B in a fullyassembled state;

FIGS. 45A & 45B are oppositely facing simplified partially pictorial,partially sectional illustrations taken along lines XLVA-XLVA andXLVB-XLVB, respectively, in FIGS. 43A & 43B;

FIGS. 46A, 46B & 46C are three sectional illustrations showing theoperative orientation of the SEM compatible sample container of FIGS.41A-45B at three stages of operation;

FIG. 47 is a simplified sectional and pictorial illustrations of tissuecontaining samples and insertion into a SEM using the SEM compatiblesample container of FIGS. 41A-46C;

FIGS. 48A, 49B, 48C and 48D are simplified sectional illustrationsshowing the operative orientation of a SEM compatible sample containerat various stages of operation and insertion into a SEM using the SEMcompatible sample container constructed and operative in accordance withanother preferred embodiment of the present invention;

FIG. 49 is a simplified pictorial and sectional illustration of a SEMinspection of a sample using the SEM compatible sample container ofFIGS. 41A-46C;

FIG. 50 is a greatly enlarged simplified schematic illustration of theSEM inspection of a sample in the context of FIG. 49;

FIGS. 51A, 51B and 51C are simplified illustrations of a microscopymulti-sample holder in use with a SEM compatible sample dish of the typeshown in FIGS. 31A-40;

FIGS. 52A, 52B and 52C are simplified illustrations of a microscopymulti-sample holder in use with a SEM compatible sample dish of the typeshown in FIGS. 41A-50;

FIGS. 53A and 53B are simplified illustrations of a microscopymulti-sample holder defining a plurality of SEM compatible samplecontainers in accordance with a preferred embodiment of the presentinvention;

FIGS. 54A and 54B are simplified illustrations of a microscopymulti-sample holder defining a plurality of SEM compatible samplecontainers in accordance with a preferred embodiment of the presentinvention;

FIG. 55 is a simplified illustration of a SEM based sample inspectionsystem constructed and operative in accordance with a preferredembodiment of the present invention;

FIG. 56 is a simplified illustration of a SEM based sample inspectionsystem constructed and operative in accordance with another preferredembodiment of the present invention;

FIG. 57 is a simplified illustration of a SEM based sample inspectionsystem constructed and operative in accordance with yet anotherpreferred embodiment of the present invention;

FIGS. 58A & 58B are oppositely facing simplified exploded view pictorialillustrations of a disassembled SEM compatible sample containerconstructed and operative in accordance with yet another preferredembodiment of the present invention;

FIGS. 59A & 59B are oppositely facing simplified partially pictorial,partially sectional illustrations of a subassembly of the container ofFIGS. 58A & 58B;

FIGS. 60A & 60B are oppositely facing simplified exploded view pictorialillustrations of the SEM compatible sample container of FIGS. 58A-59B ina partially assembled state;

FIGS. 61A & 61B are oppositely facing simplified pictorial illustrationsof the SEM compatible sample container of FIGS. 58A-60B in a fullyassembled state;

FIGS. 62A & 62B are oppositely facing simplified partially pictorial,partially sectional illustrations taken along lines LXIIA-LXIIA andLXIIB-LXIIB, respectively, in FIGS. 60A & 60B;

FIGS. 63A, 63B & 63C are three sectional illustrations showing theoperative orientation of the SEM compatible sample container of FIGS.58A-62B at three stages of operation;

FIGS. 64A, 64B, 64C, 64D and 64E are simplified sectional illustrationsof cell growth, liquid removal, liquid addition, sealing and insertioninto a SEM respectively using the SEM compatible sample container ofFIGS. 58A-63C;

FIGS. 65A, 65B and 65C are simplified sectional illustrations of liquidcontaining samples, sealing and insertion into a SEM respectively usingthe SEM compatible sample container of FIGS. 58A-63C;

FIG. 66 is a simplified pictorial and sectional illustration of a SEMinspection of a sample using the SEM compatible sample container ofFIGS. 58A-63C;

FIG. 67 is a greatly enlarged simplified schematic illustration of theSEM inspection of a sample in the context of FIG. 66;

FIGS. 68A & 68B are oppositely facing simplified exploded view pictorialillustrations of a disassembled SEM compatible sample containerconstructed and operative in accordance with another preferredembodiment of the present invention;

FIGS. 69A & 69B are oppositely facing simplified partially pictorial,partially sectional illustrations of a subassembly of the container ofFIGS. 68A & 68B;

FIGS. 70A & 70B are oppositely facing simplified exploded view pictorialillustrations of the SEM compatible sample container of FIGS. 68A-69B ina partially assembled state;

FIGS. 71A & 71B are oppositely facing simplified pictorial illustrationsof the SEM compatible sample container of FIGS. 68A-70B in a fullyassembled state;

FIGS. 72A & 72B are oppositely facing simplified partially pictorial,partially sectional illustrations taken along lines LXXIIA-LXXIIA andLXXIB-LXXIIB, respectively, in FIGS. 70A & 70B;

FIGS. 73A, 73B & 73C are three sectional illustrations showing theoperative orientation of the SEM compatible sample container of FIGS.68A-72B at three stages of operation;

FIGS. 74A, 74B, 74C, 74D and 74E are simplified sectional illustrationsof cell growth, liquid removal, liquid addition, sealing and insertioninto a SEM respectively using the SEM compatible sample container ofFIGS. 68A-73C;

FIGS. 75A, 75B and 75C are simplified sectional illustrations of liquidcontaining samples, sealing and insertion into a SEM respectively usingthe SEM compatible sample container of FIGS. 68A-73C;

FIG. 76 is a simplified pictorial and sectional illustration of a SEMinspection of a sample using the SEM compatible sample container ofFIGS. 68A-73C;

FIG. 77 is a greatly enlarged simplified schematic illustration of theSEM inspection of a sample in the context of FIG. 76;

FIGS. 78A, 78B and 78C are simplified illustrations of a microscopymulti-sample holder in use with a SEM compatible sample dish of the typeshown in FIGS. 58A-67;

FIGS. 79A, 79B and 79C are simplified illustrations of a microscopymulti-sample holder in use with a SEM compatible sample dish of the typeshown in FIGS. 68A-77;

FIGS. 80A and 80B are simplified illustrations of a microscopymulti-sample holder defining a plurality of SEM compatible samplecontainers in accordance with a preferred embodiment of the presentinvention;

FIGS. 81A and 81B are simplified illustrations of a microscopymulti-sample holder defining a plurality of SEM compatible samplecontainers in accordance with a preferred embodiment of the presentinvention;

FIG. 82 is a simplified illustration of a SEM based sample inspectionsystem constructed and operative in accordance with a preferredembodiment of the present invention;

FIG. 83 is a simplified illustration of a SEM based sample inspectionsystem constructed and operative in accordance with another preferredembodiment of the present invention;

FIG. 84 is a simplified illustration of a SEM based sample inspectionsystem constructed and operative in accordance with yet anotherpreferred embodiment of the present invention;

FIG. 85 is a simplified partially pictorial and partially sectionalillustration of SEM inspection of a sample constructed and operative inaccordance with another preferred embodiment of the present invention;

FIGS. 86A and 86B are simplified partially pictorial and partiallysectional illustration of a tissue sample slicing assembly constructedand operative in accordance with a preferred embodiment of the presentinvention;

FIG. 87A and 87B are simplified partially pictorial and partiallysectional illustration of a tissue sample slicing assembly constructedand operative in accordance with a preferred embodiment of the presentinvention;

FIG. 88 is a schematic depiction of the main steps that comprise amethod for electron microscopic inspections of wet biological andenvironmental samples at a non-vacuum environment in accordance with apreferred embodiment of the present invention;

FIG. 89 is a SEM micrograph of a cultured Chinese Hamster Ovary (CHO)cell prepared and imaged in accordance with a preferred embodiment ofthe present invention;

FIGS. 90A and 90B are SEM micrographs of HeLa cells prepared and imagedin accordance with a preferred embodiment of the present invention;

FIGS. 91A and 91B are SEM micrographs, at two different magnifications,of A431 cells prepared and imaged in accordance with a preferredembodiment of the present invention;

FIG. 92 is a SEM mnicrograph of HeLa cells prepared and imaged inaccordance with a preferred embodiment of the present invention;

FIGS. 93A, 93B and 93C are SEM micrographs of HeLa cells prepared andimaged in accordance with another preferred embodiment of the presentinvention;

FIGS. 94A and 94B are SEM micrographs of Escherichia coli bacteria andBacillus subtillis bacteria, respectively, prepared and imaged inaccordance with yet another preferred embodiment of the presentinvention;

FIGS. 95A, 95B and 95C are SEM micrographs, taken at different energylevels of a scanning electron beam, of a CHO cell prepared and imaged inaccordance with still another preferred embodiment of the presentinvention;

FIGS. 96A and 96B are SEM micrographs, at two different magnifications,of a fragment of murine heart prepared and imaged in accordance with apreferred embodiment of the present invention;

FIGS. 97A, 97B, 97C and 97D are SEM micrographs of a fragment of porcineadipose tissue, prepared and imaged in accordance with another preferredembodiment of the present invention;

FIGS. 98A and 98B are SEM micrographs at two different magnifications ofretinal pigment epithelium (RPE) of a rabbit's eye, prepared and imagedin accordance with yet another preferred embodiment of the presentinvention;

FIGS. 99A and 99B are SEM micrographs at two different magnifications ofa spinal chord of a rat, prepared and imaged in accordance with stillanother preferred embodiment of the present invention;

FIGS. 100A and 100B are SEM micrographs, at different energy levels ofthe scanning electron beam of a fragment of murine pancreas, preparedand imaged in accordance with a preferred embodiment of the presentinvention;

FIGS. 101A, 101B and 101C are SEM micrographs of fragments of murinepancreas, rat tail, and mouse duodenum, respectively, prepared andimaged in accordance with another preferred embodiment of the presentinvention;

FIGS. 102A, 102B, 102C and 102D are SEM micrographs of fragments ofmurine kidney, prepared and imaged in accordance with yet anotherpreferred embodiment of the present invention;

FIGS. 103A and 103B are SEM micrographs, at two differentmagnifications, of rat cardiac muscle, prepared and imaged in accordancewith still another preferred embodiment of the present invention;

FIGS. 104A and 104B are SEM micrographs, at two differentmagnifications, of human thyroid, prepared and imaged in accordance witha preferred embodiment of the present invention;

FIG. 105 is a SEM micrograph of rat thymus, prepared and imaged inaccordance with another preferred embodiment of the present invention;

FIGS. 106A and 106B are SEM micrographs of immunolabeled rat kidneyprepared and imaged in accordance with yet another preferred embodimentof the present invention;

FIGS. 107A and 107B are SEM micrographs, at two differentmagnifications, of commercial 1.5% fat cow's milk, prepared and imagedin accordance with still another preferred embodiment of the presentinvention;

FIGS. 101A and 108B are SEM micrographs, at two differentmagnifications, of fresh human milk, prepared and imaged in accordancewith a preferred embodiment of the present invention;

FIG. 109 is a SEM micrograph of crystals of pyroantimonate saltsprepared and imaged in accordance with another preferred embodiment ofthe present invention;

FIGS. 110A and 110B are micrographs of CHO cells obtained bybackscattered electron detection and light detection, respectively, in ascanning electron microscope, in accordance with yet another preferredembodiment of the present invention;

FIGS. 111A and 111B are micrographs of Fluorescent beads obtained bybackscattered electron detection and light detection, respectively, in ascanning electron microscope, in accordance with still another preferredembodiment of the present invention;

FIGS. 112A and 112B are micrographs of SEM inspection of samples usingX-ray detection in accordance with a preferred embodiment of the presentinvention;

FIG. 113 is a schematic depiction of a method for examining patients inaccordance with a preferred embodiment of the present invention;

FIG. 114 is a schematic depiction of a method for testing the effects ofa treatment on experimental animals in accordance with another preferredembodiment of the present invention;

FIG. 115 is a schematic depiction of manufacturing process that includesSEM inspection in accordance with yet another preferred embodiment ofthe present invention; and

FIG. 116 is a schematic depiction of a method for bioassayingpharmaceutical entities or suspected or known toxic entities, inaccordance with still another preferred embodiment of the presentinvention.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The present invention relates to methods for electron microscopicinspections of wet biological and environmental samples at a non-vacuumenvironment. More specifically, the patent relates to methods forvisualizing samples in a scanning electron microscope (SEM) without theneed for dehydration procedures including water replacement andcritical-point drying, which can destroy important structural detail andintroduce artifacts in the sample to be observed. Absent the methods ofthe present invention, samples to be examined in an electron microscopemust be held in a vacuum or a near vacuum to permit unimpeded access tothe electron beam, which can only travel in a vacuum or near vacuum.

The methods of the present invention advantageously employ a novel SEMsample container, described hereinbelow, into which the sample to bescanned is placed. The sample's hydration and atmospheric pressure stateis maintained therein, even after placement on the SEM stage and the SEMscanning chamber evacuated.

Reference is now made to FIGS. 1A-5B, which are oppositely facingsimplified exploded view pictorial illustrations of a disassembledscanning electron microscope (SEM) compatible sample containerconstructed and operative in accordance with a preferred embodiment ofthe present invention. As seen in FIGS. 1A & 1B, the SEM compatiblesample container comprises first and second mutually threaded enclosureelements, respectively designated by reference numerals 100 and 102,arranged for enhanced ease and speed of closure. Enclosure elements 100and 102 are preferably molded of plastic and coated with a conductivemetal coating.

First enclosure element 100 preferably defines a liquid sample enclosureand has a base surface 104 having a generally central aperture 106. Anelectron beam permeable, fluid impermeable, membrane subassembly 108,shown in detail in FIGS. 2A and 2B, is seated inside enclosure element100 against and over aperture 106, as shown in FIGS. 3A & 3B and 5A &5B. A sample dish comprising subassembly 108 suitably positioned inenclosure element 100 is designated by reference numeral 109, as shownin FIGS. 3A-5B.

Turning additionally to FIGS. 2A and 2B, it is seen that an electronbeam permeable, fluid impermeable, membrane 110, preferably a polyimidemembrane, such as Catalog No. LWN00033, commercially available fromMoxtek Inc. of Orem, Utah, U.S.A., is adhered, as by an adhesive, to amechanically supporting grid 112. Grid 112, which is not shown to scale,is preferably Catalog No. BM 0090-01, commercially available fromBuckbee-Mears of Cortland, N.Y., U.S.A, and the adhesive is preferablyCatalog No. NOA61, commercially available from Norland Products Inc. ofCranbury, N.J., U.S.A. liquid sample enclosure defining ring 114 isadhered to electron beam permeable, fluid impermeable, membrane 110,preferably by an adhesive, such as Catalog No. NOA61, commerciallyavailable from Norland Products Inc. of Cranbury, N.J., U.S.A. Ring 114is preferably formed of PMMA (polymethyl methacrylate), such as CatalogNo. 692106001000, commercially available from Irpen of Barcelona, Spain,and preferably defines a liquid sample enclosure with a volume ofapproximately 20 microliters and a height of approximately 2 mm.Preferably ring 114 is configured to define a liquid sample enclosure116 having inclined walls.

Alternatively, membrane 110 may be formed from polyamide,polyamide-imide, polyethylene, polypyrrole, PARLODION, COLLODION,KAPTON, FORMVAR, VINYLEC, BUTVAR, PIOLOFORM, PARYLENE, silicon -dioxide,silicon monoxide or carbon, or any combination thereof or any othersuitable material.

An O-ring 118 is preferably disposed between ring 114 and an interiorsurface 120 of second enclosure element 102. O-ring 118 is operative,when enclosure elements 100 and 102 are in tight threaded engagement, toobviate the need for the threaded engagement of elements 100 and 102 tobe a sealed engagement.

Second enclosure element 102 preferably is formed with a generallycentral stub 122, which is arranged to be seated in a suitable recess(not shown) in a specimen stage of a scanning electron microscope. It isa particular feature of the present invention that the container, shownin FIGS. 1A-10, is sized and operative with conventional stub recessesin conventional scanning electron microscopes and does not require anymodification thereof whatsoever. It is appreciated that variousconfigurations and sizes of stubs may be provided so as to fit variousscanning electron microscopes.

Enclosure elements 100 and 102 are preferably also provided withrespective radially extending positioning and retaining protrusions 124and 125, to enable the container to be readily seated in a suitablemulti-container holder and also to assist users in threadably openingand closing the enclosure elements 100 and 102. Preferably, the mutualazimuthal positioning of the protrusions 124 and 125 on respectiveenclosure elements 100 and 102 is such that mutual azimuthal alignmenttherebetween indicates a desired degree of threaded closuretherebetween, as shown in FIGS. 4A and 4B.

It is appreciated that in another embodiment of the present inventionthe sample dish may include enclosures 100 and 102.

Reference is now made to FIGS. 6A, 6B & 6C, which are three sectionalillustrations showing the operative orientation of the SEM compatiblesample container of FIGS. 1A-5B at three stages of operation. FIG. 6Ashows the container of FIGS. 1A-5B containing a liquid sample 130 andarranged in the orientation shown in FIG. 1B, prior to threaded closureenclosure elements 100 and 102. It is noted that the liquid sample doesnot flow out of the liquid sample enclosure 116 due to surface tension.The electron beam permeable, fluid impermeable, membrane 110 is seen inFIG. 6A to be generally planar.

FIG. 6B shows the container of FIG. 6A immediately following fullthreaded engagement between enclosure elements 100 and 102, producingsealing of the liquid sample enclosure 116 from the ambient. It is seenthat the electron beam permeable, fluid impermeable, membrane 110 andits supporting grid 112 bow outwardly due to pressure buildup in theliquid sample enclosure 116 as the result of sealing thereof in thismanner.

FIG. 6C illustrates the container of FIG. 6B, when placed in anevacuated environment of a SEM, typically at a vacuum of 10⁻²-10⁶millibars. It is seen that in this environment, the electron beampermeable, fluid impermeable, membrane 110 and support grid 112 bowoutwardly to a greater extent than in the ambient environment of FIG. 6Band further that the electron beam permeable, fluid impermeable,membrane 110 tends to be forced into and through the interstices of grid112 to a greater extent than occurs in the ambient environment of FIG.6B.

Reference is now made to FIGS. 7A, 7B, 7C, 7D and 7E, which aresimplified sectional illustrations of cell growth, liquid removal,liquid addition, sealing and insertion into a SEM respectively using theSEM compatible sample container of FIGS. 1A-6C. Turning to FIG. 7A,which illustrates a typical cell culture situation, it is seen that theenclosure element 100 having disposed therewithin subassembly 108 is inthe orientation shown in FIG. 1A and cells 140 in a liquid medium 142are located within liquid sample enclosure 116, the cells 140 lyingagainst the electron beam permeable, fluid impermeable, membrane 110.

FIG. 7B shows removal of liquid from liquid sample enclosure 116,typically by aspiration, and FIG. 7C shows addition of liquid to liquidsample enclosure 116. It is appreciated that multiple occurrences ofliquid removal and addition may take place with respect to a samplewithin liquid sample enclosure 116. Preferably, the apparatus employedfor liquid removal and addition is designed or equipped such as toprevent inadvertent rupture of the electron beam permeable, fluidimpermeable, membrane 110.

FIG. 7D illustrates closing of the container containing the cells 140,seen in FIG. 7C, in a liquid medium 142. FIG. 7E shows the closedcontainer, in the orientation of FIG. 1B being inserted onto a stage 144of a SEM 146. It is appreciated that there exist SEMs wherein theorientation of the container is opposite to that shown in FIG. 7E.

FIGS. 7A-7D exemplify a situation wherein at least a portion of a liquidcontaining sample remains in contact with the electron beam permeable,fluid impermeable, membrane 110 notwithstanding the addition or removalof liquid from liquid sample enclosure 116. This situation may includesituations wherein part of the sample is adsorbed or otherwise adheredto the electron beam permeable, fluid impermeable, membrane 110.Examples of liquid containing samples may include cell cultures, blood,bacteria and acellular material.

Reference is now made to FIGS. 8A, 8B and 8C which are simplifiedsectional illustrations of liquid containing samples in contact with theelectron beam permeable, fluid impermeable, membrane 110, sealing andinsertion into a SEM respectively using the SEM compatible samplecontainer of FIGS. 1A-6C. FIGS. 8A-8C exemplify a situation wherein atleast a portion of a liquid containing sample 160 is in contact with theelectron beam permeable, fluid impermeable, membrane 110 but is notadhered thereto. Examples of liquid containing samples may includevarious emulsions and suspensions such as milk, cosmetic creams, paints,inks, and pharmaceuticals in liquid form. It is seen that the enclosureelement 100 in FIGS. 8A-8B, having disposed therewithin subassembly 108,is in the orientation shown in FIG. 1A.

FIG. 8B illustrates closing of the container containing the sample 160.FIG. 8C shows the closed container, in the orientation of FIG. 1B, beinginserted onto a stage 144 of a SEM 146. It is appreciated that thereexist SEMs wherein the orientation of the container is opposite to thatshown in FIG. 8C.

Reference is now made to FIG. 9, which is a simplified pictorial andsectional illustration of SEM inspection of a sample using the SEMcompatible sample container of FIGS. 1A-6C. As seen in FIG. 9, thecontainer, here designated by reference numeral 170, is shown positionedon stage 144 of a SEM 146 such that an electron beam 172, generated bythe SEM, passes through electron beam permeable, fluid impermeable,membrane 110 and impinges on a liquid containing sample 174 withincontainer 170. Backscattered electrons from sample 174 pass throughelectron beam permeable, fluid impermeable, membrane 110 and aredetected by a detector 176, forming part of the SEM. One or moreadditional detectors, such as a secondary electron detector 178 may alsobe provided. An X-ray detector (not shown) may also be provided fordetecting X-ray radiation emitted by the sample 174 due to electron beamexcitation thereof.

Reference is now made additionally to FIG. 10, which schematicallyillustrates some details of the electron beam interaction with thesample 174 in container 170 in accordance with a preferred embodiment ofthe present invention. It is noted that the present invention enableshigh contrast imaging of features which are distinguished from eachother by their average atomic number, as illustrated in FIG. 10. In FIG.10 it is seen that nucleoli 180, having a relatively high average atomicnumber, backscatter electrons more than the surrounding nucleoplasm 182.

It is also noted that in accordance with a preferred embodiment of thepresent invention, imaging of the interior of the sample to a depth ofup to approximately 2 microns is achievable for electrons having anenergy level of less than 50 KeV, as seen in FIG. 10, wherein nucleoli180 disposed below electron beam permeable, fluid impermeable, membrane110 are imaged.

Reference is now made to FIGS. 11A-15B, which are oppositely facingsimplified exploded view pictorial illustrations of a disassembledscanning electron microscope (SEM) compatible sample containerconstructed and operative in accordance with another preferredembodiment of the present invention. As seen in FIGS. 11A & 11B, the SEMcompatible sample container comprises first and second mutually threadedenclosure elements, respectively designated by reference numerals 200and 202, arranged for enhanced ease and speed of closure. Enclosureelements 200 and 202 are preferably molded of plastic and coated with aconductive metal coating.

First enclosure element 200 preferably defines a liquid sample enclosureand has a base surface 204 having a generally central aperture 206. Anelectron beam permeable, fluid impermeable, membrane subassembly 208,shown in detail in FIGS. 12A and 12B, is seated inside enclosure element200 against and over aperture 206, as shown in FIGS. 13A & 13B and 15A &15B. A sample dish comprising subassembly 208 suitably positioned inenclosure element 200 is designated by reference numeral 209, as shownin FIGS. 13A-15B.

Turning additionally to FIGS. 12A and 12B, it is seen that an electronbeam permeable, fluid impermeable, membrane 210, preferably a polyimidemembrane, such as Catalog No. LWN00033, commercially available fromMoxtek Inc. of Orem, Utah, U.S.A., is adhered, as by an adhesive, to amechanically supporting grid 212. Grid 212, which is not shown to scale,is preferably Catalog No. BM 0090-01, commercially available fromBuckbee-Mears of Cortland, N.Y., U.S.A., and the adhesive is preferablyCatalog No. NOA61, commercially available from Noiland Products Inc. ofCranbury, N.J., U.S.A. A liquid sample enclosure defining ring 214 isadhered to electron beam permeable, fluid impermeable, membrane 210,preferably by an adhesive, such as Catalog No NOA61, commerciallyavailable from Norland Products Inc. of Cranbury, N.J., U.S.A. Ring 214is preferably formed of PMMA (polymethyl methacrylate), such as CatalogNo. 692106001000, commercially available from Irpen of Barcelona, Spain,and preferably defines a liquid sample enclosure with a volume ofapproximately 20 microliters and a height of approximately 2 mm.Preferably ring 214 is configured to define a liquid sample enclosure216 having inclined walls.

A diaphragm 218 is preferably disposed between ring 214 and an interiorsurface 219 of second enclosure element 202. Diaphragm 218 is preferablyintegrally formed of an O-ring portion 220 to which is sealed anexpandable sheet portion 221. The diaphragm 218 is preferably molded ofsilicon rubber having a Shore hardness of about 50 and the sheet portion221 preferably has a thickness of 0.2-0.3 mm. Diaphragm 218 isoperative, when enclosure elements 200 and 202 are in tight threadedengagement, to obviate the need for the threaded engagement of elements200 and 202 to be a sealed engagement and to provide dynamic and staticpressure relief.

Second enclosure element 202 preferably is formed with a generallycentral stub 222, having a throughgoing bore 223, which stub is arrangedto be seated in a suitable recess (not shown) in a specimen stage of ascanning electron microscope. Bore 223 enables diaphragm 218 to providepressure relief by defining a fluid communication channel between oneside of the diaphragm 218 and the environment in which the (SEM)compatible sample container is located. It is a particular feature ofthe present invention that the container, shown in FIGS. 11A-20, issized and operative with conventional stub recesses in conventionalscanning electron microscopes and does not require any modificationthereof whatsoever. It is appreciated that various configurations andsizes of stubs may be provided so as to fit various scanning electronmicroscopes.

Enclosure elements 200 and 202 are preferably also provided withrespective radially extending positioning and retaining protrusions 224and 225, to enable the container to be readily seated in a suitablemulti-container holder and also to assist users in threadably openingand closing the enclosure elements 200 and 202. Preferably, the mutualazimuthal positioning of the protrusions 224 and 225 on respectiveenclosure elements 200 and 202 is such that mutual azimuthal alignmenttherebetween indicates a desired degree of threaded closuretherebetween, as shown in FIGS. 14A and 14B.

Reference is now made to FIGS. 16A, 16B & 16C, which are three sectionalillustrations showing the operative orientation of the SEM compatiblesample container of FIGS. 11A-15B at three stages of operation. FIG. 16Ashows the container of FIGS. 11A-15B containing a liquid sample 230 andarranged in the orientation shown in FIG. 11B, prior to threaded closureenclosure elements 200 and 202. It is noted that the liquid sample doesnot flow out of the liquid sample enclosure 216 due to surface tension.The electron beam permeable, fluid impermeable, membrane 210 is seen inFIG. 16A to be generally planar.

FIG. 16B shows the container of FIG. 16A immediately following fullthreaded engagement between enclosure elements 200 and 202, producingsealing of the liquid sample enclosure 216 from the ambient. It is seenthat the diaphragm 218 bows outwardly due to pressure buildup in theliquid sample enclosure 216 as the result of sealing thereof in thismanner. In this embodiment, electron beam permeable, fluid impermeable,membrane 210 and its supporting grid 212 also bow outwardly due topressure buildup in the liquid sample enclosure 216 as the result ofsealing thereof in this manner, however to a significantly lesserextent, due to the action of diaphragm 218. This can be seen bycomparing FIG. 16B with FIG. 6B.

FIG. 16C illustrates the container of FIG. 16B, when placed in anevacuated environment of a SEM, typically at a vacuum of 10⁻²-10⁻⁶millibars. It is seen that in this environment, the diaphragm 218 bowsoutwardly to a greater extent than in the ambient environment of FIG.16B and that electron beam permeable, fluid impermeable, membrane 210and support grid 212 also bow outwardly to a greater extent than in theambient environment of FIG. 16B, but to a significantly lesser extentthan in the embodiment of FIG. 6C, due to the action of diaphragm 218.This can be seen by comparing FIG. 16C with FIG. 6C.

It is also noted that the electron beam permeable, fluid impermeable,membrane 210 tends to be forced into and through the interstices of grid212 to a greater extent than occurs in the ambient environment of FIG.16B but to a significantly lesser extent than in the embodiment of FIG.6C, due to the action of diaphragm 218. This can also be seen bycomparing FIG. 16C with FIG. 6C.

Reference is now made to FIGS. 17A, 17B, 17C, 17D and 17E, which aresimplified sectional illustrations of cell growth, liquid removal,liquid addition, sealing and insertion into a SEM respectively using theSEM compatible sample container of FIGS. 11A-16C. Turning to FIG. 17A,which is identical to FIG. 7A and illustrates a typical cell culturesituation, it is seen that the enclosure element 200 having disposedtherewithin subassembly 208 is in the orientation shown in FIG. 11A andcells 240 in a liquid medium 242 are located within liquid sampleenclosure 216, the cells 240 lying against the electron beam permeable,fluid impermeable, membrane 210.

FIG. 17B, which is identical to FIG. 7B, shows removal of liquid fromliquid sample enclosure 216, typically by aspiration, and FIG. 17C,which is identical to FIG. 7C, shows addition of liquid to liquid sampleenclosure 216. It is appreciated that multiple occurrences of liquidremoval and addition may take place with respect to a sample withinliquid sample enclosure 216. Preferably, the apparatus employed forliquid removal and addition is designed or equipped such as to preventinadvertent rupture of the electron beam permeable, fluid impermeable,membrane 210.

FIG. 17D illustrates closing of the container containing the cells 240,seen in FIG. 17C, in a liquid medium 242. FIG. 17 shows the closedcontainer, in the orientation of FIG. 11B being inserted onto a stage244 of a SEM 246. It is appreciated that there exist SEMs wherein theorientation of the container is opposite to that shown in FIG. 17E.

FIGS. 17A-17D exemplify a situation wherein at least a portion of aliquid containing sample remains in contact with the electron beampermeable, fluid impermeable, membrane 210 notwithstanding the additionor removal of liquid from liquid sample enclosure 216. This situationmay include situations wherein part of the sample is adsorbed orotherwise adhered to the electron beam permeable, fluid impermeable,membrane 210. Examples of liquid containing samples may include cellcultures, blood, bacteria and acellular material.

Reference is now made to FIGS. 18A, 18B and 18C which are simplifiedsectional illustrations of liquid containing samples in contact with theelectron beam permeable, fluid impermeable, membrane 210, sealing andinsertion into a SEM respectively using the SEM compatible samplecontainer of FIGS. 11A-16C. FIGS. 18A-18C exemplify a situation whereinat least a portion of a liquid containing sample 260 is in contact withthe electron beam permeable, fluid impermeable, membrane 210 but is notadhered thereto. Examples of liquid containing samples may includevarious emulsions and suspensions such as milk, cosmetic creams, paints,inks, and pharmaceuticals in liquid form. It is seen that the enclosureelement 200, having disposed therewithin subassembly 208 in FIGS. 8A-8B,is in the orientation shown in FIG. 11A. FIG. 18A is identical to FIG.8A.

FIG. 18B illustrates closing of the container containing the sample 260.FIG. 18C shows the closed container, in the orientation of FIG. 11B,being inserted onto a stage 244 of a SEM 246. It is appreciated thatthere exist SEMs wherein the orientation of the container is opposite tothat shown in FIG. 18C.

Reference is now made to FIG. 19, which is a simplified pictorial andsectional illustration of SEM inspection of a sample using the SEMcompatible sample container of FIGS. 11A-16C. As seen in FIG. 19, thecontainer, here designated by reference numeral 270, is shown positionedon stage 244 of a SEM 246 such that an electron beam 272, generated bythe SEM, passes through electron beam permeable, fluid impermeable,membrane 210 and impinges on a liquid containing sample 274 withincontainer 270. Backscattered electrons from sample 274 pass throughelectron beam permeable, fluid impermeable, membrane 210 and aredetected by a detector 276, forming part of the SEM. One or moreadditional detectors, such as a secondary electron detector 278 may alsobe provided. An X-ray detector (not shown) may also be provided fordetecting X-ray radiation emitted by the sample 274 due to electron beamexcitation thereof.

Reference is now made additionally to FIG. 20, which schematicallyillustrates some details of the electron beam interaction with thesample 274 in container 270 in accordance with a preferred embodiment ofthe present invention. It is noted that the present invention enableshigh contrast imaging of features which are distinguished from eachother by their average atomic number, as illustrated in FIG. 20. In FIG.20 it is seen that nucleoli 280 having a relatively high average atomicnumber, backscatter electrons more than the surrounding nucleoplasm 282.

It is also noted that in accordance with a preferred embodiment of thepresent invention, imaging of the interior of the sample to a depth ofup to approximately 2 microns is achievable for electrons having anenergy level of less than 50 KeV, as seen in FIG. 20, wherein nucleoli280 disposed below electron beam permeable, fluid impermeable, membrane210 are imaged.

Reference is now made to FIGS. 21A and 21B, which are simplifiedexploded view illustrations of a pre-microscopy multi-sample holder inuse with SEM compatible sample containers of the type shown in FIGS.1A-20 and to FIGS. 22A and 22B which are simplified illustrations of thepre-microscopy multi-sample holder of FIGS. 21A & 21B respectivelyuncovered and covered in an assembled state.

As seen in FIGS. 21A and 21B, the pre-microscopy multi-sample holderpreferably comprises a base 300, a top element 302 and a cover 304,Cover 304 is preferably provided to maintain sterility within theinterior of the pre-microscopy multi-sample holder.

The base 300 is preferably injection molded of a plastic material anddefines an array of container support locations 306. Each containersupport location 306 is preferably defined by a recess 308 having alight transparent bottom wall through which light microscopy may takeplace. Adjacent to each recess 308 there is preferably formed a pair ofmutually aligned pairs of upstanding mutually spaced protrusions 310arranged to receive protrusions, designated by reference numeral 124 inFIGS. 1A-4B, on enclosure elements, designated by reference numeral 100in FIGS. 1A-8C, thereby fixing the azimuthal alignment thereof.

Base 300 preferably also defines a plurality of liquid reservoirs 312which are adapted to hold liquid used to maintain a desired level ofhumidity in the interior of the pre-microscopy multi-sample holder. Base300 is preferably formed with a floor 320. Top element 302 is arrangedfor removable snap-fit engagement with base 300 so as to retain sampledishes, designated by reference numeral 109 in FIGS. 3A-5B, in a desiredarray on base 300. Top element 302 is formed with a planar surface 322having an array of apertures 324 which are arranged to overlie thesample dishes 109 when seated at container support locations 306. Thesize of apertures 324 is preferably selected to be less than the size ofthe enclosure elements 100, so as to prevent the sample dishes 109 frompassing therethrough. Planar surface 322 preferably also includesapertures 326 communicating with liquid reservoirs 312.

Top element 302 also provides positioning guides 328 and dummy apertures330 for use by a suction device in conjunction therewith, as describedhereinbelow with reference to FIG. 23A and 23B. Combination dummyapertures 332 are also provided. Only part of each aperture 332 covers aliquid reservoir 312, while the reminder of each aperture 332 serves asa dummy aperture for a suction device.

Cover 304 is provided to maintain sterility of the interior of thepre-microscopy multi-sample holder. Cover 304 is preferably transparentto light, as illustrated in FIG. 22B. The pre-microscopy multi-sampleholder of FIGS. 21A-22B is preferably dimensioned so as to be compatiblewith conventional cell biology equipment, such as light microscopes,centrifuges and automated positioning devices, Preferred dimensions are85 nm×127 mm.

Reference is now made to FIGS. 23A, 23B and 23C, which are simplifiedillustrations of the pre-microscopy multi-sample holder of FIGS. 21A-22Brespectively associated with a suction device and pipettes. Turning toFIG. 23A, it is seen that the suction device, here designated byreference numeral 350, comprises a manifold 352 coupled via a conduit354 to a source of suction. The manifold 352 preferably communicateswith a linear array of uniformly spaced needles 356. A pair of spacers358 is attached to the manifold 352 or is integrally formed therewith.Spacers 358 are arranged in line with the linear array of needles 356.These spacers 358 preferably engage floor 320 of base 300 atintermediate adjacent positioning guides 328 on opposite sides of thetop element 302. The spacers 358 ensure that the needles 356 do notengage electron beam permeable, fluid impermeable, membrane, designatedby reference numeral 110 in FIGS. 1A-10.

As seen in FIG. 23A, the container support locations 306 are arranged instaggered rows on the pre-microscopy multi-sample holder. Thus, as seenin FIG. 23B, in every row, three of the needles 356 engage apertures324, two of the needles 356 engage dummy apertures 330 and one of theneedles 356 engages the part of an aperture 332 which serves as a dummyaperture.

FIG. 23C illustrates addition of liquid to individual sample dishes 109by means of conventional pipettes 360. Collar elements 362 may beprovided for use in association with pipettes 360 to prevent inadvertentengagement of the pipettes with electron beam permeable, fluidimpermeable, membrane, designated by reference numeral 110 in FIGS.1A-10.

Reference is now made to FIGS. 24A, 24B and 24C, which are simplifiedillustrations of a microscopy multi-sample holder in use with a SEMcompatible sample dish of the type shown in FIGS. 1A-10. As seen in FIG.24A, the microscopy multi-sample holder preferably comprises a base 400and a sealing cover 404. The base 400 is preferably injection molded ofa plastic material and defines an array of dish support locations 406.Each dish support location 406 is preferably defined by an aperture 408through which SEM microscopy may take place. Adjacent to each aperture408 there is preferably formed a pair of mutually aligned pairs ofupstanding mutually spaced protrusions 410 arranged to receiveprotrusions 424 on sample dishes 425. Sample dishes 425 may be generallyidentical to sample dishes 109, shown in FIGS. 3A -5B, but do notrequire any threading or other attachment mechanism.

Base 400 may define a plurality of liquid reservoirs 412 which areadapted to hold liquid used to maintain a desired level of humidity inthe interior of the microscopy multi-sample holder.

Sealing cover 404 is arranged for individual sealing engagement witheach of sample dishes 425. Preferably sealing cover 404 is provided onthe underside thereof with an array of O-rings 426, shown in FIG. 24C,sealed thereto and arranged so as to sealingly engage a top rim surfaceof each of sample dishes 425, when the sealing cover 404 is in place,preferably in removable snap-fit engagement with base 400.

FIG. 24B shows the apparatus of FIG. 24A with one sample dish 425positioned at a dish support location 406 in base 400. FIG. 24C showssealing cover 404 in snap fit engagement with base 400, therebyproviding individual sealing of each of sample dishes 425 by means ofO-ring 426 and a portion of sealing cover 404 circumscribed thereby.

Reference is now made to FIGS. 25A, 25B and 25C, which are simplifiedillustrations of a microscopy multi-sample holder in use with a SEMcompatible sample dish of the type shown in FIGS. 11A-20. As seen inFIG. 25A, the microscopy multi-sample holder preferably comprises a base450 and a sealing cover 454. The base 450 is preferably injection moldedof a plastic material and defines an array of dish support locations456. Each dish support location 456 is preferably defined by an aperture458 through which SEM microscopy may take place. Adjacent to eachaperture 458 there is preferably formed a pair of mutually aligned pairsof upstanding mutually spaced protrusions 460 arranged to receiveprotrusions 474 on sample dishes 475. Sample dishes 475 may be generallyidentical to sample dishes 209, shown in FIGS. 13A-15B, but do notrequire any threading or other attachment mechanism.

Base 450 may define a plurality of liquid reservoirs 462 which areadapted to hold liquid used to maintain a desired level of humidity inthe interior of the microscopy multi-sample holder.

Sealing cover 454 is arranged for individual sealing engagement of eachof sample dishes 475 with a diaphragm 476, shown in FIG. 25C, which issealingly mounted over an aperture 478 formed in sealing cover 454.Preferably an array of diaphragms 476, which may be identical todiaphragms 218 described hereinabove with reference to FIGS. 11A-20, isprovided on the underside of sealing cover 454. The individualdiaphragms 476 are arranged so as to sealingly engage a top rim surfaceof each of sample dishes 475, when the sealing cover 454 is in place,preferably in removable snap-fit engagement with base 450.

FIG. 25B shows the apparatus of FIG. 25A with one sample dish 475positioned at a dish support location 456 in base 450. FIG. 25C showssealing cover 454 in snap fit engagement-with base 450, therebyproviding individual sealing of each of sample dishes 475 by means ofdiaphragm 476.

Reference is now made to FIGS. 26A and 26B, which are simplifiedillustrations of a microscopy multi-sample holder defining a pluralityof SEM compatible sample containers in accordance with a preferredembodiment of the present invention. As seen in FIG. 26A, the microscopymulti-sample holder preferably comprises a base 500 and a sealing cover504. The base 500 is preferably injection molded of a plastic materialand defines an array of sample containers 506. Each sample container 506preferably includes an aperture 508 through which SEM microscopy maytake place. An electron beam permeable, fluid impermeable, membrane 510,shown in FIG. 26B, is sealed over each aperture 508. Membrane 510 ispreferably identical to membrane 110 described hereinabove withreference to FIGS. 1A-10. Sealing cover 504 preferably is arranged forindividual sealing engagement with each of sample containers 506.

FIG. 26B shows the apparatus of FIG. 26A in sealed engagement, therebyproviding individual sealing of each of sample containers 506.

Reference is now made to FIGS. 27A and 27B, which are simplifiedillustrations of a microscopy multi-sample holder defining a pluralityof SEM compatible sample containers in accordance with a preferredembodiment of the present invention As seen in FIG. 27A, the microscopymulti-sample holder preferably comprises a base 550 and a sealing cover554. The base 550 is preferably injection molded of a plastic materialand defines an array of sample containers 556. Each sample container 556preferably includes an aperture 558 through which SEM microscopy maytake place. An electron beam permeable, fluid impermeable, membrane 560,shown in FIG. 27B, is sealed over each aperture 558. Membrane 560 ispreferably identical to membrane 210 described hereinabove withreference to FIGS. 11A-20. Sealing cover 554, preferably a diaphragmformed of resilient sheet material such as silicon rubber of 0.2-0.3 mmin thickness and having a Shore hardness of about 50, is arranged forindividual sealing engagement with each of sample containers 556.

FIG. 27B shows the apparatus of FIG. 27A in sealed engagement, therebyproviding individual sealing of each of sample containers 556.

Reference is now made to FIG. 28, which is a simplified illustration ofa SEM based sample inspection system constructed and operative inaccordance with a preferred embodiment of the present invention. As seenin FIG. 28, a plurality of pre-microscopy multi-sample holders 600, eachcontaining a multiplicity of SEM compatible sample containers 602 of thetype shown in FIGS. 1A-20, is shown in an incubator 604. Preferably,light microscopy inspection of the samples in containers 602 is carriedout while the containers 602 are mounted in holder 600, as indicated atreference numeral 606, in order to identify samples of interest.Preferably an inverted light microscope 608 is employed for thispurpose.

Preferably automated positioning systems, such as robotic arms, asshown, are used for conveying the pre-microscopy multi-sample holders600 and the containers 602 throughout the system, it being appreciatedthat manual intervention may be employed at one or more stages asappropriate.

Thereafter, individual containers 602 are removed from holders 600 andplaced on a removable electron microscope specimen stage 610, which issubsequently introduced into a scanning electron microscope 612. Theresulting image may be inspected visually by an operator and/or analyzedby conventional image analysis functionality, typically embodied in acomputer 614.

Reference is now made to FIG. 29, which is a simplified illustration ofa SEM based sample inspection system constructed and operative inaccordance with another preferred embodiment of the present invention.As seen in FIG. 29, a plurality of microscopy multi-sample holders 650,each containing a multiplicity of SEM compatible sample dishes 652 ofeither of the types shown in FIGS. 24A-25C, is shown in an incubator654. Preferably, light microscopy inspection of the samples in sampledishes 652 is carried out while the sample dishes are mounted in holder650, as indicated at reference numeral 656, in order to identify samplesof interest. Preferably an inverted light microscope 658 is employed forthis purpose.

Preferably automated positioning systems, such as robotic arms, asshown, are used for conveying the microscopy multi-sample holders 650containing sample dishes 652 throughout the system, it beingappreciated-that manual intervention may be employed at one or morestages as appropriate.

Thereafter, holders 650 are placed on an electron microscope specimenstage 660, which is subsequently introduced into a scanning electronmicroscope 662. The resulting images may be inspected visually by anoperator and/or analyzed by conventional image analysis functionality,typically embodied in a computer 664.

Reference is now made to FIG. 30, which is a simplified illustration ofa SEM based sample inspection system constructed and operative inaccordance with yet another preferred embodiment of the presentinvention. As seen in FIG. 30, a plurality of microscopy multi-sampleholders 670, each defining a multiplicity of SEM compatible samplecontainers 672, as shown in any of FIGS. 26A-27B, is seen in anincubator 674. Preferably, light microscopy inspection of the samples insample containers 672 is carried out holder-wise, as indicated atreference numeral 676, preferably in order to identify samples ofinterest. Preferably an inverted light microscope 678 is employed forthis purpose.

Preferably automated positioning systems, such as robotic arms, asshown, are used for conveying the microscopy multi-sample holders 670throughout the system, it being appreciated that manual intervention maybe employed at one or more stages as appropriate.

Thereafter, holders 670 are placed on an electron microscope specimenstage 680, which is subsequently introduced into a scanning electronmicroscope 682. The resulting images may be inspected visually by anoperator and/or analyzed by conventional image analysis functionality,typically embodied in a computer 684.

Reference is now made to FIGS. 31A-35B, which are oppositely facingsimplified exploded view pictorial illustrations of a disassembledscanning electron microscope (SEM) compatible sample containerconstructed and operative in accordance with another preferredembodiment of the present invention. As seen in FIGS. 31A & 31B, the SEMcompatible sample container comprises first and second threadedenclosure elements, respectively designated by reference numerals 1100and 1102, arranged for enhanced ease and speed of closure. Enclosureelements 1100 and 1102 are preferably molded of plastic and coated witha conductive metal coating.

First enclosure element 1100 preferably defines a sample enclosure andhas a base surface 1104 having a generally central aperture 1106. Anelectron beam permeable, fluid impermeable, membrane subassembly 1108,shown in detail in FIGS. 32A and 32B, is seated inside enclosure element1100 against and over aperture 1106, as shown in FIGS. 33A & 33B and 35A& 35B. A sample dish comprising subassembly 1108 suitably positioned inenclosure element 1100 is designated by reference numeral 1109, as shownin FIGS. 33A-35B.

Turning additionally to FIGS. 32A and 32B, it is seen that an electronbeam permeable, fluid impermeable, membrane 1110, preferably a polyimidemembrane, such as Catalog No. LWN00033, commercially available fromMoxtek Inc. of Orem, Utah, U.S.A., is adhered, as by an adhesive, to amechanically supporting grid 1112. Grid 1112, which is not shown toscale, is preferably Catalog No. BM 0090-01, commercially available fromBuckbee-Mears of Cortland, N.Y., U.S.A., and the adhesive is preferablyCatalog No. NOA61, commercially available from Norland Products Inc. ofCranbury, N.J., U.S.A. A sample enclosure defining ring 1114 is adheredto electron beam permeable, fluid impermeable, membrane 1110, preferablyby an adhesive, such as Catalog No. NOA61, commercially available. fromNorland Products Inc. of Cranbury, N.J., U.S.A. Ring 1114 is preferablyformed of PMMA (polymethyl methacrylate), such as Catalog No.692106001000, commercially available from Irpen of Barcelona, Spain, andpreferably defines a sample enclosure with a volume of approximately 20microliters and a height of approximately 2 mm. Preferably ring 1114 isconfigured to define a sample enclosure 1116 having inclined walls.

A first O-ring 1118 is preferably disposed between an interior surface1120 of second enclosure element 1102 and a connecting element 1122.Connecting element 1122 is preferably molded of plastic and coated witha conductive metal coating. A second O-ring 1123 is preferably disposedbetween connecting element 1122 and ring 1114 of subassembly 1108.O-rings 1118 and 1123 are operative, when enclosure elements 1100 and1102 and connecting element 1122 are in tight threaded engagement, toobviate the need for the threaded engagement of elements 1100 and 1102and connecting element 1122 to be a sealed engagement.

Connecting element 1122 preferably has a recess 1124. Connecting element1122 is also formed with a protrusion 1126, seen in FIGS. 35A & 35B,protruding into recess 1124.

A positioner 1128 is preferably comprised of two upright flexibleprojections 1130, each with a ridge 1132 formed on an end 1134 of theprojections 1130. Positioner 1128 is preferably molded of plastic.Projections 1130 press against each other when inserted into recess 1124of connecting element 1122 and then snap back to an upright positiononce ridges 1132 are seated on the protrusion 1126 of connecting element1122, as shown in FIGS. 35A & 35B.

Positioner 1128 is preferably also provided with respective radiallyextending positioning and retaining protrusions 1136 extending from arim 1137. Positioning and retaining protrusions 1136 are seated inapertures 1140 formed in the inclined walls of sample enclosure 1116 ofring 1114 to prevent rotation of positioner 1128.

A coil spring 1142 is disposed on positioner 1128 between rim 1137 andridges 1132 of projections 1130. Spring 1142 is preferably formed ofhardened stainless steel.

The positioner 1128 and spring 1142 are operative to move a non-liquidsample up and against electron beam permeable, fluid impermeable,membrane 1110 when enclosure elements 1100 and 1102 and connectingelement 1122 are in tight threaded engagement.

Second enclosure element 1102 is preferably formed with a generallycentral stub 1150, which is arranged to be seated in a suitable recess(not shown) in a specimen stage of a scanning electron microscope. It isa particular feature of the present invention that the container, shownin FIGS. 31A-40, is sized and operative with conventional stub recessesin conventional scanning electron microscopes and does not require anymodification thereof whatsoever. It is appreciated that variousconfigurations and sizes of stubs may be provided so as to fit variousscanning election microscopes.

Enclosure elements 1100 and 1102 are preferably also provided withrespective radially extending positioning and retaining protrusions 1154and 1155, to enable the container to be readily seated in a suitablemulti-container holder and also to assist users in threadably openingand closing the enclosure elements 1100 and 1102. Preferably, the mutualazimuthal positioning of the protrusions 1154 and 1155 on respectiveenclosure elements 1100 and 1102 is such that mutual azimuthal alignmenttherebetween indicates a desired degree of threaded closuretherebetween, as shown in FIGS. 34A and 34B.

Reference is now made to FIGS. 36A, 36B & 36C, which are three sectionalillustrations showing the operative orientation of the SEM compatiblesample container of FIGS. 31A-35B at three stages of operation. FIG. 36Ashows the container of FIGS. 31A-35B containing a tissue sample 1160 andarranged in the orientation shown in FIG. 31B, prior to threaded closureof enclosure elements 1100 and 1102 and connecting element 1122. Theelectron beam permeable, fluid impermeable, membrane 1110 is seen inFIG. 36A to be generally planar.

FIG. 36B shows the container of FIG. 36A immediately following fullthreaded engagement between enclosure elements 1100 and 1102 andconnecting element 1122 producing sealing of the tissue sample enclosure1116 from the ambient. It is noted that the tissue sample 1160 is inclose contact with the electron beam permeable, fluid impermeable,membrane 1110 due to the force exerted by the positioner 1128. It isseen that the electron beam permeable, fluid impermeable, membrane 1110and its supporting grid 1112 bow outwardly due to pressure buildup inthe tissue sample enclosure 1116 as the result of sealing thereof inthis manner.

FIG. 36C illustrates the container of FIG. 36B, when placed in anevacuated environment of a SEM, typically at a vacuum of 10⁻²-10⁻⁶millibars. It is seen that in this environment, the electron beampermeable, fluid impermeable, membrane 1110 and support grid 1112 bowoutwardly to a greater extent than in the ambient environment of FIG.36B and further that the electron beam permeable, fluid impermeable,membrane 1110 tends to be forced into and through the interstices ofgrid 1112 to a greater extent than occurs in the ambient environment ofFIG. 36B.

Reference is now made to FIG. 37, which is a simplified sectional andpictorial illustration of tissue containing sample and insertion into aSEM using the SEM compatible sample container of FIGS. 31A-36C.

FIG. 37 shows the closed container, in the orientation of FIG. 31B,being inserted onto a stage 1164 of a SEM 1166. It is appreciated thatthere exist SEMs wherein the orientation of the container is opposite tothat shown in FIG. 37.

Reference is now made to FIGS. 38A, 38B, 38C and 38D, which are foursectional illustrations showing the operative orientation of a variationof the SEM compatible sample container of FIGS. 31A-35B at four stagesof operation. FIG. 38A shows a container 1170, identical to thecontainer of FIGS. 31A-35B other than as specified hereinbelow,containing a sample including cells 1172 grown on a cell growth platform1174 and arranged in the orientation shown in FIG. 31B, prior tothreaded closure of enclosure elements 1100 and 1102 and connectingelement 1122. The electron beam permeable, fluid impermeable, membrane1110 is seen in FIG. 38A to be generally planar. Cell growth platform1174 is removably mounted onto a suitably configured positioner 1176,which corresponds to positioner 1128 in the embodiment of FIGS. 31A-37.Typically, the cells are grown onto cell growth platform 1174 whileplatform 1174 is not mounted onto positioner 1176. The mounting ofplatform 1174 onto positioner 1176 typically occurs just before SEMinspection takes place.

FIG. 38B shows the container of FIG. 38A immediately following fullthreaded engagement between enclosure elements 1100 and 1102 andconnecting element 1122 producing sealing of the cell sample enclosure,here designated by reference numeral 1178, from the ambient It is notedthat the sample containing cells 1172 is in close contact with theelectron beam permeable, fluid impermeable, membrane 1110 due to theforce exerted by the positioner 1176. It is seen that the electron beampermeable, fluid impermeable, membrane 1110 and its supporting grid 1112bow outwardly due to pressure buildup in the cell sample enclosure 1178as the result of sealing thereof in this manner.

FIG. 38C illustrates the container of FIG. 38B, when placed in anevacuated environment of a SEM, typically at a vacuum of 10⁻²-10⁻⁶millibars. It is seen that in this environment, the electron beampermeable, fluid impermeable, membrane 1110 and support grid 1112 bowoutwardly to a greater extent than in the ambient environment of FIG.38B and further that the electron beam permeable, fluid impermeable,membrane 1110 tends to be forced into and through the interstices ofgrid 1112 to a greater extent than occurs in the ambient environment ofFIG. 38B.

FIG. 38D shows the closed container 1170, in the orientation of FIG.31B, being inserted onto stage 1164 of SEM 1166. It is appreciated thatthere exist SEMs wherein the orientation of the container is opposite tothat shown in FIG. 38D.

Reference is now made to FIG. 39, which is a simplified pictorial andsectional illustration of SEM inspection of a sample using the SEMcompatible sample container of FIGS. 31A-37. As seen in FIG. 39, thecontainer, here designated by reference numeral 1180, is shownpositioned on stage 1164 of SEM 1166 such that an electron beam 1182,generated by the SEM, passes through electron beam permeable, fluidimpermeable, membrane 1110 and impinges on a tissue containing sample1184 within container 1180. Backscattered electrons from sample 1184pass through electron beam permeable, fluid impermeable, membrane 1110and are detected by a detector 1186, forming part of the SEM. One ormore additional detectors, such as a secondary electron detector 1188,may also be provided. An X-ray detector (not shown) may also be providedfor detecting X-ray radiation emitted by the sample 1184 due to electronbeam excitation thereof.

Reference is now made additionally to FIG. 40, which schematicallyillustrates some details of the electron beam interaction with thesample 1184 in container 1180 in accordance with a preferred embodimentof the present invention. It is noted that the present invention enableshigh contrast imaging of features which are distinguished from eachother by their average atomic number, as illustrated in FIG. 40. In FIG.40 it is seen that nucleoli 1190, having a relatively high averageatomic number, backscatter electrons more than the surroundingnucleoplasm 1192.

It is also noted that in accordance with a preferred embodiment of thepresent invention, imaging of the interior of the sample to a depth ofup to approximately 2 microns is achievable for electrons having anenergy level of less than 50 KeV, as seen in FIG. 40, wherein nucleoli1190 disposed below electron beam permeable, fluid impermeable, membrane1110 are imaged.

Reference is now made to FIGS. 41A-45B, which are oppositely facingsimplified exploded view pictorial illustrations of a disassembledscanning electron microscope (SEM) compatible sample containerconstructed and operative in accordance with another preferredembodiment of the present invention. As seen in FIGS. 41A & 41B, the SEMcompatible sample container comprises first and second threadedenclosure elements, respectively designated by reference numerals 1200and 1202, arranged for enhanced ease and speed of closure. Enclosureelements 1200 and 1202 are preferably molded of plastic and coated witha conductive metal coating.

First enclosure element 1200 preferably defines a sample enclosure andhas a base surface 1204 having a generally central aperture 1206. Anelectron beam permeable, fluid impermeable, membrane subassembly 1208,shown in detail in FIGS. 42A and 42B, is seated inside enclosure element1200 against and over aperture 1206, as shown in FIGS. 43A & 43B and 45A& 45B. A sample dish comprising subassembly 1208 suitably positioned inenclosure element 1200 is designated by reference numeral 1209, as shownin FIGS. 43A-45B.

Turning additionally to FIGS. 42A and 42B, it is seen that an electronbeam permeable, fluid impermeable, membrane 1210, preferably a polyimidemembrane, such as Catalog No. LWN00033, commercially available fromMoxtek Inc. of Orem, Utah, U.S.A., is adhered, as by an adhesive, to amechanically supporting grid 1212. Grid 1212, which is not shown toscale, is preferably Catalog No. BM 0090-01, commercially available fromBuckbee-Mears of Cortland, N.Y., U.S.A., and the adhesive is preferablyCatalog No. NOA61, commercially available from Norland Products Inc. ofCranbury, N.J., U.S.A. A sample enclosure defining ring 1214 is adheredto electron beam permeable, fluid impermeable, membrane 1210, preferablyby an adhesive, such as Catalog No. NOA61, commercially available fromNorland Products Inc. of Cranbury, N.J., U.S.A. Ring 1214 is preferablyformed of PMMA (polymethyl methacrylate), such as Catalog No.692106001000, commercially available from Irpen of Barcelona, Spain, andpreferably defines a sample enclosure with a volume of approximately 20microliters and a height of approximately 2 mm. Preferably ring 1214 isconfigured to define a sample enclosure 1216 having inclined walls.

A diaphragm 1218 is preferably integrally formed of an O-ring portion1220 to which is sealed an expandable sheet portion 1221. The diaphragm1218 is preferably disposed between an interior surface 1219 of secondenclosure element 1202 and a connecting element 1222. Connecting element1222 is preferably molded of plastic and coated with a conductive metalcoating. The diaphragm 1218 is preferably molded of silicon rubberhaving a Shore hardness of about 50 and the sheet portion 1221preferably has a thickness of 0.2-0.3 mm.

An O-ring 1223 is preferably disposed between connecting element 1222and ring 1214 of subassembly 1208. Diaphragm 1218 and O-ring 1223 areoperative, when enclosure elements 1200 and 1202 and connecting element1222 are in tight threaded engagement, to obviate the need for thethreaded engagement of elements 1200 and 1202 and connecting element1222 to be a sealed engagement and to provide dynamic and staticpressure relief.

Connecting element 1222 preferably has a central recess 1224. Connectingelement 1222 is also formed with a protrusion 1226, seen in FIGS. 45A&45B, protruding into recess 1224.

A positioner 1228 is preferably comprised of two upright flexibleprojections 1230, each with a ridge 1232 formed on an end 1234 of theprojections 1230. Positioner 1128 is preferably molded of plastic.Projections 1230 press against each other when inserted into recess 1224of connecting element 1222 and then snap back to an upright positiononce ridges 1232 are seated on the protrusion 1226 of connecting element1222, as shown in FIGS. 45A & 45B.

Positioner 1228 is preferably also provided with respective radiallyextending positioning and retaining protrusions 1236 extending from arim 1237. Positioning and retaining protrusions 1236 are seated inapertures 1240 formed in inclined walls of sample enclosure 1216 of ring1214 to prevent rotation of positioner 1228.

A coil spring 1242 is disposed on positioner 1228 between rim 1237 andridges 1232 of projections 1230. Spring 1242 is preferably formed ofhardened stainless steel.

The positioner 1228 and spring 1242 are operative to move a non-liquidsample up and against electron beam permeable, fluid impermeable,membrane 1210 when enclosure elements 1200 and 1202 and connectingelement 1222 are in tight threaded engagement.

Second enclosure element 1202 is preferably formed with a generallycentral stub 1250, having a throughgoing bore 1252, which stub isarranged to be seated in a suitable recess (not shown) in a specimenstage of a scanning electron microscope. Bore 1252 enables diaphragm1218 to provide pressure relief by defining a fluid communicationchannel between one side of the diaphragm 1218 and the environment inwhich the (SEM) compatible sample container is located. It is aparticular feature of the present invention that the container, shown inFIGS. 41A-50, is sized and operative with conventional stub recesses inconventional scanning electron microscopes and does not require anymodification thereof whatsoever. It is appreciated that variousconfigurations and sizes of stubs may be provided so as to fit variousscanning electron microscopes.

Enclosure elements 1200 and 1202 are preferably also provided withrespective radially extending positioning and retaining protrusions 1254and 1255, to enable the container to be readily seated in a suitablemulti-container holder and also to assist users in threadably openingand closing the enclosure elements 1200 and 1202. Preferably, the mutualazimuthal positioning of the protrusions 1254 and 1255 on respectiveenclosure elements 1200 and 1202 is such that mutual azimuthal alignmenttherebetween indicates a desired degree of threaded closuretherebetween, as shown in FIGS. 44A and 44B.

Reference is now made to FIGS. 46A, 46B & 46C, which are three sectionalillustrations showing the operative orientation of the SEM compatiblesample container of FIGS. 41A-45B at three stages of operation. FIG. 46Ashows the container of FIGS. 41A-45B containing a tissue sample 1260 andarranged in the orientation shown in FIG. 41B, prior to threaded closureenclosure elements 1200 and 1202 and connecting element 1222. Theelectron beam permeable, fluid impermeable, membrane 1210 is seen inFIG. 46A to be generally planar.

FIG. 46B shows the container of FIG. 46A immediately following fullthreaded engagement between enclosure elements 1200 and 1202 andconnecting, element 1222, producing sealing of the tissue sampleenclosure 1216 from the ambient It is noted that the tissue sample 1260is in close contact with the electron beam permeable, fluid impermeable,membrane 1210 due to the force exerted by the positioner 1228. It isseen that the diaphragm 1218 bows outwardly due to pressure buildup inthe tissue sample enclosure 1216 as the result of sealing thereof inthis manner. In this embodiment, electron beam permeable, fluidimpermeable, membrane 1210 and its supporting grid 1212 also bowoutwardly due to pressure buildup in the tissue sample enclosure 1216 asthe result of sealing thereof in this manner, however to a significantlylesser extent, due to the action of diaphragm 1218. This can be seen bycomparing FIG. 46B with FIG. 36B.

FIG. 46C illustrates the container of FIG. 46B, when placed in anevacuated environment of a SEM, typically at a vacuum of 10⁻²-10⁻⁶millibars. It is seen that in this environment, the diaphragm 1218 bowsoutwardly to a greater extent than in the ambient environment of FIG.46B and that electron beam permeable, fluid impermeable, membrane 1210and support grid 1212 also bow outwardly to a greater extent than in theambient environment of FIG. 46B, but to a significantly lesser extentthan in the embodiment of FIG. 36C, due to the action of diaphragm 1218.This can be seen by comparing FIG. 46C with FIG. 36C.

It is also noted that the electron beam permeable, fluid impermeable,membrane 1210 tends to be forced into and through the interstices ofgrid 1212 to a greater extent than occurs in the ambient environment ofFIG. 46B but to a significantly lesser extent than in the embodiment ofFIG. 36C, due to the action of diaphragm 1218. This can also be seen bycomparing FIG. 46C with FIG. 36C.

Reference is now made to FIG. 47, which is a simplified sectional andpictorial illustration of tissue containing samples and insertion into aSEM using the SEM compatible sample container of FIGS. 41A-46C.

FIG. 47 shows the closed container, in the orientation of FIG. 41B,being inserted onto a stage 1264 of a SEM 1266. It is appreciated thatthere exist SEMs wherein the orientation of the container is opposite tothat shown in FIG. 47.

Reference is now made to FIGS. 48A, 48B, 48C and 48D, which are foursectional illustrations showing the operative orientation of a variationof the SEM compatible sample container of FIGS. 41A-45B at four stagesof operation. FIG. 48A shows a container 1270, identical to thecontainer of FIGS. 41A-45B other than as specified hereinbelow,containing a sample including cells 1272 grown on a cell growth platform1274 and arranged in the orientation shown in FIG. 41B, prior tothreaded closure of enclosure elements 1200 and 1202 and connectingelement 1222. The electron beam permeable, fluid impermeable, membrane1210 is seen in FIG. 48A to be generally planar. Cell growth platform1274 is removably mounted onto a suitably configured positioner 1276,which corresponds to positioner 1228 in the embodiment of FIGS. 41A-47.Typically, the cells are grown onto cell growth platform 1274 whileplatform 1274 is not mounted onto positioner 1276. The mounting ofplatform 1274 onto positioner 1276 typically occurs just before SEMinspection takes place.

FIG. 48B shows the container of FIG. 48A immediately following fullthreaded engagement between enclosure elements 1200 and 1202 andconnecting element 1222 producing sealing of the cell sample enclosure,here designated by reference numeral 1278, from the ambient It is notedthat the sample containing cells 1272 is in close contact with theelectron beam permeable, fluid impermeable, membrane 1210 due to theforce exerted by the positioner 1276. It is seen that the electron beampermeable, fluid impermeable, membrane 1210 and its supporting grid 1212bow outwardly due to pressure buildup in the cell sample enclosure 1278as the result of sealing thereof in this manner, however to asignificantly lesser extent than in the embodiment of FIG. 38B, due tothe action of diaphragm 1218. This can be seen by comparing FIG. 48Bwith FIG. 38B.

FIG. 48C illustrates the container of FIG. 48B, when placed in anevacuated environment of a SEM, typically at a vacuum of 10⁻²-10⁻⁶millibars. It is seen that in this environment, the electron beampermeable, fluid impermeable, membrane 1210 and support grid 1212 bowoutwardly to a greater extent than in the ambient environment of FIG.48B and further that the electron beam permeable, fluid impermeable,membrane 1210 tends to be forced into and through the interstices ofgrid 1212 to a greater extent than occurs in the ambient environment ofFIG. 48B, but to a significantly lesser extent than in the embodiment ofFIG. 38C, due to the action of diaphragm 1218. This can be seen bycomparing FIG. 48C with FIG. 38C.

FIG. 48D shows the closed container 1270, in the orientation of FIG.41B, being inserted onto stage 1264 of SEM 1266. It is appreciated thatthere exist SEMs wherein the orientation of the container is opposite tothat shown in FIG. 48D.

Reference is now made to FIG. 49, which is a simplified pictorial andsectional illustration of SEM inspection of a sample using the SEMcompatible sample container of FIGS. 41A-47. As seen in FIG. 49, thecontainer, here designated by -reference numeral 1280, is shownpositioned on stage 1264 of SEM 1266 such that an electron beam 1282,generated by the SEM, passes through electron beam permeable, fluidimpermeable, membrane 1210 and impinges on a tissue containing sample1284 within container 1280. Backscattered electrons from sample 1284pass through electron beam permeable, fluid impermeable, membrane 1210and are detected by a detector 1286, forming part of the SEM. One ormore additional detectors, such as a secondary electron detector 1288,may also be provided. An X-ray detector (not shown) may also be providedfor detecting X-ray radiation emitted by the sample 1284 due to electronbeam excitation thereof.

Reference is now made additionally to FIG. 50, which schematicallyillustrates some details of the electron beam interaction with thesample 1284 in container 1280 in accordance with a preferred embodimentof the present invention It is noted that the present invention enableshigh contrast imaging of features which are distinguished from eachother by their average atomic number, as illustrated in FIG. 50. In FIG.50 it is seen that nucleoli 1290, having a relatively high averageatomic number, backscatter electrons more than the surroundingnucleoplasm 1292.

It is also noted that in accordance with a preferred embodiment of thepresent invention, imaging of the interior of the sample to a depth ofup to approximately 2 microns is achievable for electrons having anenergy level of less than 50 KeV, as seen in FIG. 50, wherein nucleoli1290 disposed below electron beam permeable, fluid impermeable, membrane1210 are imaged.

Reference is now made to FIGS. 51A, 51B and 51C, which are simplifiedillustrations of a microscopy multi-sample holder in use with a SEMcompatible sample dish of the type shown in FIGS. 31A-40. As seen inFIG. 51A, the microscopy multi-sample holder preferably comprises a base1400 and a sealing cover 1404. The base 1400 is preferably injectionmolded of a plastic material and defines an array of dish supportlocations 1406. Each dish support location 1406 is preferably defined byan aperture 1408 through which SEM microscopy may take place. Adjacentto each aperture 1408 there is preferably formed a pair of mutuallyaligned pairs of upstanding mutually spaced protrusions 1410 arranged toreceive protrusions 1424 on sample dishes 1425. Sample dishes 1425 maybe generally identical to sample dishes 1109, shown in FIGS. 33A-35B,but do not require any threading or other attachment mechanism.

Sealing cover 1404 is preferably arranged for individual sealingengagement with each of sample dishes 1425. Preferably sealing cover1404 is provided on the underside thereof with an array of O-rings 1426,shown in FIG. 51C, sealed thereto and arranged so as to sealingly engagea top rim surface of each of sample dishes 1425, when the sealing cover1404 is in place, preferably in removable snap-fit engagement with base1400.

Preferably, sealing cover 1404 is provided on the underside thereof withan array of positioners 1428, shown in FIG. 51C, and arranged so as tomove non-liquid samples up and against electron beam permeable, fluidimpermeable, membrane 1110 (shown in FIGS. 31A-40) seated in sample dish1425. Individual positioners 1428 are suspended within coils 1430, asshown in FIG. 51C.

FIG. 51B shows the apparatus of FIG. 51A with one sample dish 1425positioned at a dish support location 1406 in base 1400. FIG. 51C showssealing cover 1404 in snap fit engagement with base 1400, therebyproviding individual sealing of each of sample dishes 1425 by means ofO-ring 1426 and a portion of sealing cover 1404 circumscribed thereby.

Reference is now made to FIGS. 52A, 52B and 52C, which are simplifiedillustrations of a microscopy multi-sample holder in use with a SEMcompatible sample dish of the type shown in FIGS. 41A-50. As seen inFIG. 52A, the microscopy multi-sample holder preferably comprises a base1450 and a sealing cover 1454. The base 1450 is preferably injectionmolded of a plastic material and defines an array of dish supportlocations 1456. Each dish support location 1456 is preferably defined byan aperture 1458 through which SEM microscopy may take place. Adjacentto each aperture 1458 there is preferably formed a pair of mutuallyaligned pairs of upstanding mutually spaced protrusions 1460 arranged toreceive protrusions 1474 on sample dishes 1475. Sample dishes 1475 maybe generally identical to sample dishes 1209, shown in FIGS. 43A-45B,but do not require any threading or other attachment mechanism.

Preferably sealing cover 1454 is provided on the underside thereof withan array of O-rings 1476, shown in FIG. 52C, sealed thereto and arrangedso as to sealingly engage a top rim surface of each of sample dishes1475, when the sealing cover 1454 is in place, preferably in removablesnap-fit engagement with base 1450.

Sealing cover 1454 is arranged for individual sealing engagement of eachof sample dishes 1475 with a diaphragm 1477, shown in FIG. 52C, which issealingly mounted over an aperture 1478 formed in sealing cover 1454.Preferably an array of diaphragms 1477, which may be identical todiaphragms 1218 described hereinabove with reference to FIGS. 41A-50, isprovided on the underside of sealing cover 1454. The individualdiaphragms 1477 are arranged so as to sealingly engage a top rim surfaceof each of sample dishes 1475, when the sealing cover 1454 is in place,preferably in removable snap-fit engagement with base 1450.

Preferably, sealing cover 1454 is provided with an array of positioners1480. Individual positioners 1480 are suspended within coils 1482, asshown in FIG. 51C, so as to move non-liquid samples up and againstelectron beam permeable, fluid impermeable, membrane 1210 (shown inFIGS. 41A-50) seated in sample dish 1475.

FIG. 52B shows the apparatus of FIG. 52A with one sample dish 1475positioned at a dish support location 1456 in base 1450. FIG. 52C showssealing cover 1454 in snap fit engagement with base 1450, therebyproviding individual sealing of each of sample dishes 1475 by means ofdiaphragm 1476.

Reference is now made to FIGS. 53A and 53B, which are simplifiedillustrations of a microscopy multi-sample holder defining a pluralityof SEM compatible sample containers in accordance with a preferredembodiment of the present invention. As seen in FIG. 53A, the microscopymulti-sample holder preferably comprises a base 1500 and a sealing cover1504. The base 1500 is preferably injection molded of a plastic materialand defines an array of sample containers 1506. Each sample container1506 preferably includes an aperture 1508 through which SEM microscopymay take place. An electron beam permeable, fluid impermeable, membrane1510, shown in FIG. 53B, is sealed over each aperture 1508. Membrane1510 is preferably identical to membrane 1110 described hereinabove withreference to FIGS. 31A-40. Sealing cover 1504 preferably is arranged forindividual sealing engagement with each of sample containers 1506.

Preferably, sealing cover 1504 is provided with an array of positioners1520, shown in FIG. 53B. Individual positioners 1520 are suspendedwithin coils 1522, as shown in FIG. 53B, so as to move non-liquidsamples up and against electron beam permeable, fluid impermeable,membrane 1510.

FIG. 53B shows the apparatus of FIG. 53A in sealed engagement, therebyproviding individual sealing of each of sample containers 1506.

Reference is now made to FIGS. 54A and 54B, which are simplifiedillustrations of a microscopy multi-sample holder defining a pluralityof SEM compatible sample containers in accordance with a preferredembodiment of the present invention. As seen in FIG. 54A, the microscopymulti-sample holder preferably comprises a base 1550 and a sealing cover1554. The base 1550 is preferably injection molded of a plastic materialand defines an array of sample containers 1556. Each sample container1556 preferably includes an aperture 1558 through which SEM microscopymay take place.

An electron beam permeable, fluid impermeable, membrane 1560, shown inFIG. 54B, is sealed over each aperture 1558. Membrane 1560 is preferablyidentical to membrane 1210 described hereinabove with reference to FIGS.41A-50. Sealing cover 1554 preferably comprises a diaphragm 1562 formedof resilient sheet material such as silicon rubber of 0.2-0.3 mm inthickness and having a Shore hardness of about 50. Diaphragm 1562 issealingly mounted over apertures 1564 formed in sealing cover 1554 andis arranged for individual sealing engagement with each of samplecontainers 1556.

Preferably, sealing cover 1554 is provided with an array of positioners1570, shown in FIG. 54B. Individual positioners 1570 are suspendedwithin coils 1572, as shown in FIG. 54B, so as to move non-liquidsamples up and against electron beam permeable, fluid impermeable,membrane 1560.

FIG. 54B shows the apparatus of FIG. 54A in sealed engagement, therebyproviding individual sealing of each of sample containers 1556.

Reference is now made to FIG. 55, which is a simplified illustration ofa SEM based sample inspection system constructed and operative inaccordance with a preferred embodiment of the present invention. As seenin FIG. 55, preferably, automated positioning systems, such as roboticarms, as shown, are used for conveying a multiplicity of SEM compatiblesample containers 1602 throughout the system, it being appreciated thatmanual intervention may be employed at one or more stages asappropriate.

Thereafter, individual containers 1602 are placed on a removableelectron microscope specimen stage 1610, which is subsequentlyintroduced into a scanning electron microscope 1612. The resulting imagemay be inspected visually by an operator and/or analyzed by conventionalimage analysis functionality, typically embodied in a computer 1614.

Reference is now made to FIG. 56, which is a simplified illustration ofa SEM based sample inspection system constructed and operative inaccordance with another preferred embodiment of the present invention.As seen in FIG. 56, a plurality of microscopy multi-sample holders 1650,each containing a multiplicity of SEM compatible sample dishes 1652 ofeither of the types shown in FIGS. 51A-52C, is shown on a table 1654.Preferably, light microscopy inspection of the samples in sample dishes1652 is carried out while the sample dishes are mounted in holder 1650,as indicated at reference numeral 1656, in order to identify samples ofinterest. Preferably a dissection microscope 1658 is employed for thispurpose.

Preferably automated positioning systems, such as robotic arms, asshown, are used for conveying the microscopy multi-sample holders 1650containing sample dishes 1652 throughout the system, it beingappreciated that manual intervention may be employed at one or morestages as appropriate.

Thereafter, holders 1650 are placed on an electron microscope specimenstage 1660, which is subsequently introduced into a scanning electronmicroscope 1662. The resulting images may be inspected visually by anoperator and/or analyzed by conventional image analysis functionality,typically embodied in a computer 1664.

Reference is now made to FIG. 57, which is a simplified illustration ofa SEM based sample inspection system constructed and operative inaccordance with yet S another preferred embodiment of the presentinvention. As seen in FIG. 57, a plurality of microscopy multi-sampleholders 1670, each defining a multiplicity of SEM compatible samplecontainers 1672, as shown in any of FIGS. 53A-54B, are seen on a table1674. Preferably, light microscopy inspection of the samples in samplecontainers 1672 is carried out holder-wise, as indicated at referencenumeral 1676, in order to identify samples of interest. Preferably adissection microscope 1678 is employed for this purpose.

Preferably automated positioning systems, such as robotic arms, asshown, are used for conveying the microscopy multi-sample holders 1670throughout the system, it being appreciated that manual intervention maybe employed at one or more stages as appropriate.

Thereafter, holders 1670 are placed on an electron microscope specimenstage 1680, which is subsequently introduced into a scanning electronmicroscope 1682. The resulting images may be inspected visually by anoperator and/or analyzed by conventional image analysis functionality,typically embodied in a computer 1684.

Reference is now made to FIGS. 58A-62B, which are oppositely facingsimplified exploded view pictorial illustrations of a disassembledscanning electron microscope (SEM) compatible sample containerconstructed and operative in accordance with yet another preferredembodiment of the present invention. As seen in FIGS. 58A & 58B, the SEMcompatible sample container comprises first and second mutually threadedenclosure elements, respectively designated by reference numerals 2100and 2102, arranged for enhanced ease and speed of closure. Enclosureelements 2100 and 2102 are preferably molded of plastic and coated witha conductive metal coating.

First enclosure element 2100 preferably defines a liquid sampleenclosure and has a base surface 2104 having a generally centralaperture 2106. An electron beam permeable, fluid impermeable, membranesubassembly 2108, shown in detail in FIGS. 59A and 59B, is seated insideenclosure element 2100 against and over aperture 2106, as shown in FIGS.60A & 60B and 62A & 62B. A sample dish comprising subassembly 2108suitably positioned in enclosure element 2100 is designated by referencenumeral 2109, as shown in FIGS. 60A-62B.

Turning additionally to FIGS. 59A and 59B, it is seen that an electronbeam permeable, fluid impermeable, membrane 2110, preferably a polyimidemembrane, such as Catalog No. LWN00033, commercially available fromMoxtek Inc. of Orem, Utah, U.S.A., is adhered, as by an adhesive, to amechanically supporting grid 2112. Grid 2112, which is not shown toscale, is preferably Catalog No. BM 0090-01, commercially available fromBuckbee-Mears of Cortland, N.Y., U.S.A., and the adhesive is preferablyCatalog No. NOA61, commercially available from Norland Products Inc. ofCranbury, N.J., U.S.A. A liquid sample enclosure defining ring 2114 isadhered to electron beam permeable, fluid impermeable, membrane 2110,preferably by an adhesive, such as Catalog No. NOA61, commerciallyavailable from Norland Products Inc. of Cranbury, N.J., U.S.A. Ring 2114is preferably formed of PMMA (polymethyl methacrylate), such as CatalogNo. 692106001000, commercially available from Irpen of Barcelona, Spain,and preferably defines a liquid sample enclosure with a volume ofapproximately 20 microliters and a height of approximately 2 mm.Preferably ring 2114 is configured to define a liquid sample enclosure2116 having inclined walls.

An O-ring 2118 is preferably disposed between ring 2114 and an interiorsurface 2120 of second enclosure element 2102. O-ring 2118 is operative,when enclosure elements 2100 and 2102 are in tight threaded engagement,to obviate the need for the threaded engagement of elements 2100 and2102 to be a sealed engagement.

Second enclosure element 2102 preferably is formed with a generallycentral stub 2122, having a throughgoing bore 2123, which stub isarranged to be seated in a suitable recess (not shown) in a specimenstage of a scanning electron microscope.

Enclosure elements 2100 and 2102 are preferably also provided withrespective radially extending positioning and retaining protrusions 2124and 2125, to enable the container to be readily seated in a suitablemulti-container holder and also to assist users in threadably openingand closing the enclosure elements 2100 and 2102. Preferably, the mutualazimuthal positioning of the protrusions 2124 and 2125 on respectiveenclosure elements 2100 and 2102 is such that mutual azimuthal alignmenttherebetween indicates a desired degree of threaded closuretherebetween, as shown in FIGS. 61A and 61B.

A light guide 2126 is provided to receive light from a sample in liquidsample enclosure 2116 during SEM inspection from a side of the samplenot facing the electron beam permeable, fluid impermeable, membrane2110.

The light guide 2126 is formed of a cylinder with a truncated conicaltip 2128 and is inserted into bore 2123 of stub 2122. Light guide 2126is preferably a plastic or glass clad light guide with a numericalaperture of 0.66, commercially available from Fiberoptics Technology,Inc. of 12 Fiber Rd., Pomfret, Conn., U.S.A. and is sealed to enclosure2102 by any conventional means, such as by an adhesive.

The wall of conical tip 2128 is configured at an angle, such that theangle is smaller than a critical angle of reflection, so as to ensurethat incident photons emitted from a sample are reflected in the lightguide 2126.

Reference is now made to FIGS. 63A, 63B & 63C, which are three sectionalillustrations showing the operative orientation of the SEM compatiblesample container of FIGS. 58A-62B at three stages of operation. FIG. 63Ashows the container of FIGS. 58A-62B containing a liquid sample 2130 andarranged in the orientation shown in FIG. 5 8B, prior to threadedclosure of enclosure elements 2100 and 2102. It is noted that the liquidsample does not flow out of the liquid sample enclosure 2116 due tosurface tension. The electron beam permeable, fluid impermeable,membrane 2110 is seen in FIG. 63A to be generally planar.

FIG. 63B shows the container of FIG. 63A immediately following fillthreaded engagement between enclosure elements 2100 and 2102, producingsealing of the liquid sample enclosure 2116 from the ambient The lightguide tip 2128 is shown to be immersed in liquid sample 2130. It is seenthat the electron beam permeable, fluid impermeable, membrane 2110 andits supporting grid 2112 bow outwardly due to pressure buildup in theliquid sample enclosure 2116 as the result of sealing thereof in thismanner.

FIG. 63C illustrates the container of FIG. 63B, when placed in anevacuated environment of a SEM, typically at a vacuum of 10⁻²-10⁻⁶millibars. It is seen that in this environment, the electron beampermeable, fluid impermeable, membrane 2110 and support grid 2112 bowoutwardly to a greater extent than in the ambient environment of FIG.63B and further that the electron beam permeable, fluid impermeable,membrane 2110 tends to be forced into and through the interstices ofgrid 2112 to a greater extent than occurs in the ambient environment ofFIG. 63B.

Reference is now made to FIGS. 64A, 64B, 64C, 64D and 64E, which aresimplified sectional illustrations of cell growth, liquid removal,liquid addition, sealing and insertion into a SEM respectively using theSEM compatible sample container of FIGS. 58A-63C. Turning to FIG. 64A,which illustrates a typical cell culture situation, it is seen that theenclosure element 2100 having disposed therewithin subassembly 2108 isin the orientation shown in FIG. 58A and cells 2140 in a liquid medium2142 are located within liquid sample enclosure 2116, the cells 2140lying against the electron beam permeable, fluid impermeable, membrane2110.

FIG. 64B shows removal of liquid from liquid sample enclosure 2116,typically by aspiration, and FIG. 64C shows addition of liquid to liquidsample enclosure 2116. It is appreciated that multiple occurrences ofliquid removal and addition may take place with respect to a samplewithin liquid sample enclosure 2116. Preferably, the apparatus employedfor liquid removal and addition is designed or equipped such as toprevent inadvertent rupture of the electron beam permeable, fluidimpermeable, membrane 2110.

FIG. 64D illustrates closing of the container containing the cells 2140,seen in FIG. 64C, in a liquid medium 2142. The light guide tip 2128 isshown to be immersed in liquid medium.2142 of the liquid sample 2140.FIG. 64E shows the closed container, in the orientation of FIG. 58Bbeing inserted onto a stage 2144 of a SEM 2146. It is appreciated thatthere exist SEMs wherein the orientation of the container is opposite tothat shown in FIG. 64E.

FIGS. 64A-64D exemplify a situation wherein at least a portion of aliquid containing sample remains in contact with the electron beampermeable, fluid impermeable, membrane 2110 notwithstanding the additionor removal of liquid from liquid sample enclosure 2116. This situationmay include situations wherein part of the sample is adsorbed orotherwise adhered to the electron beam permeable, fluid impermeable,membrane 2110. Examples of liquid containing samples may include cellcultures, blood, bacteria and acellular material.

Reference is now made to FIGS. 65A, 65B and 65C which are simplifiedsectional illustrations of liquid containing samples in contact with theelectron beam permeable, fluid impermeable, membrane 2110, sealing andinsertion into a SEM respectively, using the SEM compatible samplecontainer of FIGS. 58A-63C. FIGS. 65A-65C exemplify a situation whereinat least a portion of a liquid containing sample 2160 is in contact withthe electron beam permeable, fluid impermeable, membrane 2110 but is notadhered thereto. Examples of liquid containing samples may includevarious emulsions and suspensions such as milk, cosmetic creams, paints,inks and pharmaceuticals in liquid form. It is seen that the enclosureelement 2100 in FIGS. 65A and 65B, having disposed therewithinsubassembly 2108, is in the orientation shown in FIG. 58A.

FIG. 65B illustrates closing of the container containing the sample2160. FIG. 65C shows the closed container, in the orientation of FIG.58B, being inserted onto stage 2144 of SEM 2146. It is appreciated thatthere exist SEMs wherein the orientation of the container is opposite tothat shown in FIG. 65C.

Reference is now made to FIG. 66, which is a simplified pictorial andsectional illustration of SEM inspection with light detection of asample using the SEM compatible sample container of FIGS. 58A-63C. Asseen in FIG. 66, the container, here designated by reference numeral2170, is shown positioned on stage 2144 of SEM 2146 in a recess 2171.Stage 2144 is operative to rotate so as to enable positioning ofcontainer 2170 under an electron beam 2172 to inspect, during SEMinspection, regions of interest within a liquid containing sample 2174.

The electron beam 2172, generated by the SEM, passes through electronbeam permeable, fluid impermeable, membrane 2110 and impinges on sample2174 within container 2170. Backscattered electrons from sample 2174pass through electron beam permeable, fluid impermeable, membrane 2110and are detected by a detector 2176, forming part of the SEM. One ormore additional detectors, such as a secondary electron detector 2178,may also be provided. An X-ray detector (not shown) may also be providedfor detecting X-ray radiation emitted by the sample 2174 due to electronbeam excitation thereof.

Photons 2180, emitted from liquid sample 2174 due to electron beamexcitation, are transmitted through a first light guide, here designatedby reference numeral 2184, which is the same as light guide 2126 ofFIGS. 58A-63C, to a second light guide 2186. Second light guide 2186 isoperative to transmit the photons 2180 to a light detector, such as aPhotomultiplier Tube (PMT) 2188. Time dependent measurement of lightintensity obtained from the light detector 2188, combined withinformation on the location of the scanning electron beam, are combinedto produce an image of sample 2174 by methods known in the art,preferably as a digital image on a computer 2189.

Second light guide 2186, preferably, has a cross section with a diameterthat is larger than the diameter of the cross section of the first lightguide 2184 so as to minimize loss of photons in the passage betweenlight guides 2184 and 2186 due to refraction or to imprecise relativealignment of the two light guides.

In the illustrated embodiment, second light guide 2186 is preferablyformed in an L-shaped curve and preferably comprises a multiplicity ofoptic fibers disposed along the L-shaped light guide 2186 at an anglethat ensures internal reflection of photons 2180 throughout the lengthof second light guide 2186.

It is appreciated that in the present invention photons 2180 may betransmitted vertically downward from first light guide 2184, via secondlight guide 2186 to a light detector located beneath the floor of theSEM 2146. Alternatively, photons 2180 may be transmitted from firstlight guide 2184 to a light detector, and that second light guide 2186may be obviated.

In another embodiment of the present invention, the photons 2180 may bespectrally resolved prior to detection by light detector 2188 byconventional means, such as filters, diffraction gratings or prisms (notshown). This allows detection of photons with a wavelength within one ormore specified ranges, yielding additional information on thecomposition and structure of the sample 2174 or features within sample2174.

Additionally, a liquid, such as oil or a gel (not shown), with an indexof refraction similar to the index of refraction of light guides 2184and 2186, may be placed between light guides 2184 and 2186 to preventthe photons 2180 from scattering outside light guides 2184 and 2186.

Reference is now made additionally to FIG. 67, which schematicallyillustrates some details of the electron beam and photon interactionwith the sample. 2174 in container 2170 in accordance with a preferredembodiment of the present invention. It is noted that the presentinvention enables high contrast imaging of features which aredistinguished from each other by their average atomic number or,alternatively, by their average photon yield due to excitation byelectrons, as illustrated in FIG. 67. In FIG. 67 it is seen thatnucleoli 2190, having a relatively high average atomic number,backscatter electrons more than the surrounding nucleoplasm 2192.

Photons 2180 are shown to emit from the nucleoli 2190 and aretransmitted to the light detector 2188 (shown in FIG. 66), via lightguide 2184. It is noted that the contrast obtained by detection ofbackscattered electrons and the contrast obtained by photon detectionare due to different physical processes and to different chemicalproperties of features within the sample, and therefore do not generallyoverlap.

It is also noted that in accordance with a preferred embodiment of thepresent invention, imaging of the interior of the sample to a depth ofup to approximately 2 microns is achievable when employing electronbeams having an energy level of less than 50 KeV, as seen in FIG. 67,wherein nucleoli 2190 disposed below electron beam permeable, fluidimpermeable, membrane 2110 are imaged.

Reference is now made to FIGS. 68A-72B, which are oppositely facingsimplified exploded view pictorial illustrations of a disassembledscanning electron microscope (SEM) compatible sample containerconstructed and operative in accordance with another preferredembodiment of the present invention As seen in FIGS. 68A & 68B, the SEMcompatible sample container comprises first and second mutually threadedenclosure elements, respectively designated by reference numerals 2200and 2202, arranged for enhanced ease and speed of closure. Enclosureelements 2200 and 2202 are preferably molded of plastic and coated witha conductive metal coating.

First enclosure element 2200 preferably defines a liquid sampleenclosure and has a base surface 2204 having a generally centralaperture 2206. An electron beam permeable, fluid impermeable, membranesubassembly 2208, shown in detail in FIGS. 69A and 69B, is seated insideenclosure element 2200 against and over aperture 2206, as shown in FIGS.70A & 70B and 72A & 72B. A sample dish comprising subassembly 2208suitably positioned in enclosure element 2200 is designated by referencenumeral 2209, as shown in FIGS. 70A-72B.

Turning additionally to FIGS. 69A and 69B, it is seen that an electronbeam permeable, fluid impermeable, membrane 2210, preferably a polyimidemembrane, such as Catalog No. LWN00033, commercially available fromMoxtek Inc. of Orem, Utah, U.S.A., is adhered, as by an adhesive, to amechanically supporting grid 2212. Grid 2212, which is not shown toscale, is preferably Catalog No. BM 0090-01, commercially available fromBuckbee-Mears of Cortland, N.Y., U.S.A., and the adhesive is preferablyCatalog No. NOA61, commercially available from Norland Products Inc. ofCranbury, N.J., U.S.A. A liquid sample enclosure defining ring 2214 isadhered to electron beam permeable, fluid impermeable, membrane 2210,preferably by an adhesive, such as Catalog No. NOA61, commerciallyavailable from Norland Products Inc. of Cranbury, N.J., U.S.A. Ring 2214is preferably formed of PMMA (polymethyl methacrylate), such as CatalogNo. 692106001000, commercially available from Irpen of Barcelona, Spain,and preferably defines a liquid sample enclosure with a volume ofapproximately 20 microliters and a height of approximately 2 mm.Preferably ring 2214 is configured to define a liquid sample enclosure2216 having inclined walls.

A diaphragm 2218 is preferably provided and is operative to providedynamic and static pressure relief. Diaphragm 2218 is preferablyintegrally formed of an O-ring portion 2220 to which is sealed anexpandable sheet portion 2221. The diaphragm 2218 is preferably moldedof silicon rubber having a Shore hardness of about 50 and the sheetportion 2221 preferably has a thickness of 0.2-0.3 mm.

The diaphragm 2218 is inserted into a first aperture 2222 formed in anexterior surface of a wall of the second enclosure element 2202. Asecond aperture 2223, shown in FIGS. 72A&72B, is formed in an interiorsurface of a wall of the second enclosure element 2202. First aperture2222 and second aperture 2223 enable diaphragm 2218 to provide pressurerelief by defining a fluid communication channel between one side of thediaphragm 2218 and the environment in which the SEM compatible samplecontainer is located. A plug 2224 is preferably provided to retain thediaphragm 2218 in aperture 2222. Plug 2224 is preferably formed of aring 2225 having a generally central aperture 2226 and is attached to aninternal surface of second enclosure element 2202 defined by aperture2222 by any conventional means, such as by a tight fitting engagement.

An O-ring 2228 is preferably disposed between ring 2214 and an interiorsurface 2230 of second enclosure element 2202. O-ring 2228 is operative,when enclosure elements 2200 and 2202 are in tight threaded engagement,to obviate the need for the threaded engagement of elements 2200 and2202 to be a sealed engagement.

Second Enclosure element 2202 preferably is formed with a generallycentral stub 2232, having a throughgoing bore 2233, which stub isarranged to be seated in a suitable recess (not shown) in a specimenstage of a scanning electron microscope.

Enclosure elements 2200 and 2202 are preferably also provided withrespective radially extending positioning and retaining protrusions 2234and 2235, to enable the container to be readily seated in a suitablemulti-container holder and also to assist users in threadably openingand closing the enclosure elements 2200 and 2202. Preferably, the mutualazimuthal positioning of the protrusions 2234 and 2235 on respectiveenclosure elements 2200 and 2202 is such that mutual azimuthal alignmenttherebetween indicates a desired degree of threaded closuretherebetween, as shown in FIGS. 71A and 71B.

A light guide 2236 is provided to receive light from a sample in liquidsample enclosure 2216 during SEM inspection from a side of the samplenot facing the electron beam permeable, fluid impermeable, membrane2210.

Light guide 2236 is formed of a cylinder with a truncated conical tip2238 and is inserted into bore 2233 of stub 2232. Light guide 2236 ispreferably a plastic or glass clad light guide with a numerical apertureof 0.66, commercially available from Fiberoptics Technology, Inc. of 12Fiber Rd., Pomfret, Conn., U.S.A and is sealed to enclosure 2202 by anyconventional means, such as by an adhesive.

The wall of conical tip 2238 is configured at an angle, such that theangle is smaller than a critical angle of reflection, so as to ensurethat incident photons emitted from a sample are reflected in the lightguide 2236.

Reference is now made to FIGS. 73A, 73B & 73C, which are three sectionalillustrations showing the operative orientation of the SEM compatiblesample container of FIGS. 68A-72B at three stages of operation. FIG. 73Ashows the container of FIGS. 68A-72B containing a liquid sample 2239 andarranged in the orientation shown in FIG. 68B, prior to threaded closureof enclosure elements 2200 and 2202. It is noted that the liquid sampledoes not flow out of the liquid sample enclosure 2216 due to surfacetension. The electron beam permeable, fluid impermeable, membrane 2210is seen in FIG. 73A to be generally planar.

FIG. 73B shows the container of FIG. 73A immediately following fullthreaded engagement between enclosure elements 2200 and 2202, producingsealing of the liquid sample enclosure 2216 from the ambient The lightguide tip 2238 is shown to be immersed in liquid sample 2239. It is seenthat the diaphragm 2218 bows outwardly due to pressure buildup in theliquid sample enclosure 2216 as the result of sealing thereof in thismanner. In this embodiment, electron beam permeable, fluid impermeable,membrane 2210 and its supporting grid 2212 also bow outwardly due topressure buildup in the liquid sample enclosure 2216 as the result ofsealing thereof in this manner, however to a significantly lesser extentthan in the embodiment of FIG. 63B, due to the action of diaphragm 2218.This can be seen by comparing FIG. 73B with FIG. 63B.

FIG. 73C illustrates the container of FIG. 73B, when placed in anevacuated environment of a SEM, typically at a vacuum of 10⁻²-10⁻⁶millibars. It is seen that in this environment, the diaphragm 2218 bowsoutwardly to a greater extent than in the ambient environment of FIG.73B and that electron beam permeable, fluid impermeable, membrane 2210and support grid 2212 also bow outwardly to a greater extent than in theambient environment of FIG. 73B, but to a significantly lesser extentthan in the embodiment of FIG. 63C, due to the action of diaphragm 2218.This can be seen by comparing FIG. 73C with FIG. 63C.

It is also noted that the electron beam permeable, fluid impermeable,membrane 2210 tends to be forced into and through the interstices ofgrid 2212 to a greater extent than occurs in the ambient environment ofFIG. 73B but to a significantly lesser extent than in the embodiment ofFIG. 63C, due to the action of diaphragm 2218. This can also be seen bycomparing FIG. 73C with FIG. 63C.

Reference is now made to FIGS. 74A, 74B, 74C, 74D and 74E, which aresimplified sectional illustrations of cell growth, liquid removal,liquid addition, sealing and insertion into a SEM, respectively, usingthe SEM compatible sample container of FIGS. 68A-73C. Turning to FIG.74A, which is identical to FIG. 64A and illustrates a typical cellculture situation, it is seen that the enclosure element 2200 havingdisposed therewithin subassembly 2208 is in the orientation shown inFIG. 68A and cells 2240 in a liquid medium 2242 are located withinliquid sample enclosure 2216, the cells 2240 lying against the electronbeam permeable, fluid impermeable, membrane 2210.

FIG. 74B, which is identical to FIG. 64B, shows removal of liquid fromliquid sample enclosure 2216, typically by aspiration, and FIG. 74C,which is identical to FIG. 64C, shows addition of liquid to liquidsample enclosure 2216. It is appreciated that multiple occurrences ofliquid removal and addition may take place with respect to a samplewithin liquid sample enclosure 2216. Preferably, the apparatus employedfor liquid removal and addition is designed or equipped such as toprevent inadvertent rupture of the electron beam permeable, fluidimpermeable, membrane 2210.

FIG. 74D illustrates closing of the container containing the cells 2240,seen in FIG. 74C, in a liquid medium 2242. The light guide tip 2238 isshown to be immersed in liquid medium 2242 of the liquid sample. FIG.74E shows the closed container, in the orientation of FIG. 68B, beinginserted onto a stage 2244 of a SEM 2246. It is appreciated that thereexist SEMs wherein the orientation of the container is opposite to thatshown in FIG. 74E.

FIGS. 74A-74D exemplify a situation wherein at least a portion of aliquid containing sample remains in contact with the electron beampermeable, fluid impermeable, membrane 2210 notwithstanding the additionor removal of liquid from liquid sample enclosure 2216. This situationmay include situations wherein part of the sample is adsorbed orotherwise adhered to the electron beam permeable, fluid impermeable,membrane 2210. Examples of liquid containing samples may include cellcultures, blood, bacteria and acellular material.

Reference is now made to FIGS. 75A, 75B and 75C which are simplifiedsectional illustrations of liquid containing samples in contact with theelectron beam permeable, fluid impermeable, membrane 2210, sealing andinsertion into a SEM, respectively, using the SEM compatible samplecontainer of FIGS. 68A-73C. FIGS. 75A-75C exemplify a situation whereinat least a portion of a liquid containing sample 2260 is in contact withthe electron beam permeable, fluid impermeable, membrane 2210 but is notadhered thereto. Examples of liquid containing samples may includevarious emulsions and suspensions such as milk, cosmetic creams, paints,inks and pharmaceuticals in liquid form It is seen that the enclosureelement 2200 in FIGS. 75A and 75B, having disposed therewithinsubassembly 2208, is in the orientation shown in FIG. 68A. FIG. 75A isidentical to FIG. 65A.

FIG. 75B illustrates closing of the container containing the sample2260. FIG. 75C shows the closed container, in the orientation of FIG.68B, being inserted onto stage 2244 of SEM 2246. It is appreciated thatthere exist SEMs wherein the orientation of the container is opposite tothat shown in FIG. 75C.

Reference is now made to FIG. 76, which is a simplified pictorial andsectional illustration of SEM inspection with light detection of asample using the SEM compatible sample container of FIGS. 68A-73C. Asseen in FIG. 76, the container, here designated by reference numeral2270, is shown positioned on stage 2244 of SEM 2246 in a recess 2271.Stage 2244 is operative to rotate so as to enable positioning ofcontainer 2270 under an electron beam 2272 to inspect, during SEMinspection, regions of interest within a liquid containing sample 2274.

The electron beam 2272, generated by the SEM, passes through electronbeam permeable, fluid impermeable, membrane 2210 and impinges on sample2274 within container 2270. Backscattered electrons from sample 2274pass through electron beam permeable, fluid impermeable, membrane 2210and are detected by a detector 2276, forming part of the SEM. One ormore additional detectors, such as a secondary electron detector 2278,may also be provided. An X-ray detector (not shown) may also be providedfor detecting X-ray radiation emitted by the sample 2274 due to electronbeam excitation thereof:

Photons 2280, emitted from liquid sample 2274 due to electron beamexcitation, are transmitted through a first light guide, here designatedby reference numeral 2284, which is identical to light guide 2226 ofFIGS. 68A-73C, to a second light guide 2286. Second light guide 2286 isoperative to transmit the photons 2280 to a light detector, such as aPhotomultiplier Tube (PMT) 2288, such as Catalog No. H6180-01,commercially available at Hamamatsu Photonics of 325-6, Sunayama-cho,Hamamatsu City, Shizuoka Pref. Japan. Time dependent measurement oflight intensity obtained from the light detector 2288, combined withinformation on the location of the scanning electron beam, are combinedto produce an image of sample 2274 by methods known in the art,preferably as a digital image on a computer 2289.

Second light guide 2286, preferably, has a cross section with a diameterthat is larger than the diameter of the cross section of the first lightguide 2284, so as to minimize loss of photons in the passage betweenlight guides 2284 and 2286 due to refraction or to imprecise relativealignment of the two light guides.

In the illustrated embodiment, second light guide 2286 is preferablyformed in an L-shaped curve and preferably comprises a multiplicity ofoptic fibers disposed along the L-shaped light guide 2286 at an anglethat ensures internal reflection of photons 2280 throughout the lengthof second light guide 2286.

It is appreciated that in the present invention photons 2280 may betransmitted vertically downward from first light guide 2284, via secondlight guide 2286 to a light detector located beneath the floor of theSEM 2246. Alternatively, photons 2280 may be transmitted from firstlight guide 2284 to a light detector, and that second light guide 2286may be obviated.

In another embodiment of the present invention, the photons 2280 may bespectrally resolved prior to detection by light detector 2288 byconventional means, such as filters, diffraction gratings or prisms (notshown). This allows detection of photons with a wavelength within one ormore specified ranges, yielding additional information on thecomposition and structure of the sample 2274 or features within sample2274.

Additionally, a liquid, such as oil or a gel (not shown), with an indexof refraction similar to the index of refraction of light guides 2284and 2286, may be placed between light guides 2284 and 2286 to preventthe photons 2280 from scattering outside light guides 2284 and 2286.

Reference is now made additionally to FIG. 77, which schematicallyillustrates some details of the electron beam and photon interactionwith the sample 2274 in container 2270 in accordance with a preferredembodiment of the present invention. It is noted that the presentinvention enables high contrast imaging of features which aredistinguished from each other by their average atomic number, or,alternatively, by their average photon yield due to excitation byelectrons, as illustrated in FIG. 77. In FIG. 77 it is seen thatnucleoli 2290, having a relatively high average atomic number,backscatter electrons more than the surrounding nucleoplasm 2292.

Photons 2280 are shown to emit from the nucleoli 2290 and aretransmitted to the light detector 2288 (shown in FIG. 76), via lightguide 2284. It is noted that the contrast obtained by detection ofbackscattered electrons and the contrast obtained by photon detectionare due to different physical processes and to different chemicalproperties of features within the sample, and therefore do not generallyoverlap.

It is also noted that in accordance with a preferred embodiment of thepresent invention, imaging of the interior of the sample to a depth ofup to approximately 2 microns is achievable when employing electronbeams having an energy level of less than 50 KeV, as seen in FIG. 77,wherein nucleoli 2290 disposed below electron beam permeable, fluidimpermeable, membrane 2210 are imaged.

It is appreciated that the pre-microscopy multi-sample holder shownhereinabove in FIGS. 21A-22B may be provided for use with SEM compatiblesample containers of the type shown in FIGS. 58A-77. Additionally, thepre-microscopy multi-sample holder may be associated with a suctiondevice and pipettes shown hereinabove in FIGS. 23A, 23B and 23C.

Reference is now made to FIGS. 78A, 78B and 78C, which are simplifiedillustrations of a microscopy multi-sample holder in use with a SEMcompatible sample dish of the type shown in FIGS. 58A-67. As seen inFIG. 78A, the microscopy multi-sample holder preferably comprises a base2400 and a sealing cover 2404. The base 2400 is preferably injectionmolded of a plastic material and defines an array of dish supportlocations 2406. Each dish support location 2406 is preferably defined byan aperture 2408 through which SEM microscopy may take place. Adjacentto each aperture 2408 there is preferably formed a pair of mutuallyaligned pairs of upstanding mutually spaced protrusions 2410 arranged toreceive protrusions 2424 on sample dishes 2425. Sample dishes 2425 maybe generally identical to sample dishes 2109, shown in FIGS. 60A -62B,but do not require any threading or other attachment mechanism.

Base 2400 preferably also defines a plurality of liquid reservoirs 2412which are adapted to hold liquid used to maintain a desired level ofhumidity in the interior of the microscopy multi-sample holder.

Sealing cover 2404 is preferably arranged for individual sealingengagement with each of sample dishes 2425. Preferably sealing cover2404 is provided on the underside thereof with an array of O-rings 2426,shown in FIG. 78C, sealed thereto and arranged so as to sealingly engagea top rim surface of each of sample dishes 2425, when the sealing cover2404 is in place, preferably in removable snap-fit engagement with base2400.

Sealing cover 2404 is preferably provided with an array of light guides2430 arranged to receive light from a sample in sample dish 2425 duringlight detection. Light guides 2430 are inserted in apertures 2432 formedon sealing cover 2404.

FIG. 78B shows the apparatus of FIG. 78A with one sample dish 2425positioned at a dish support location 2406 in base 2400. FIG. 78C showssealing cover 2404 in snap fit engagement with base 2400, therebyproviding individual sealing of each of sample dishes 2425 by means ofO-ring 2426 and a portion of sealing cover 2404 circumscribed thereby.

Reference is now made to FIGS. 79A, 79B and 79C, which are simplifiedillustrations of a microscopy multi-sample holder in use with a SEMcompatible sample dish of the type shown in FIGS. 68A-77. As seen inFIG. 79A, the microscopy multi-sample holder preferably comprises a base2450 and a sealing cover 2454. The base 2450 is preferably injectionmolded of a plastic material and defines an array of dish supportlocations 2456. Each dish support location 2456 is preferably defined byan aperture 2458 through which SEM microscopy may take place. Adjacentto each aperture 2458 there is preferably formed a pair of mutuallyaligned pairs of upstanding mutually spaced protrusions 2460 arranged toreceive protrusions 2474 on sample dishes 2475. Sample dishes 2475 maybe generally identical to sample dishes 2209, shown in FIGS. 70A-72B,but do not require any threading or other attachment mechanism.

Base 2450 preferably also defines a plurality of liquid reservoirs 2462which are adapted to hold liquid used to maintain a desired level ofhumidity in the interior of the microscopy multi-sample holder.

Sealing cover 2454 is preferably arranged for individual sealingengagement of each of sample dishes 2475. Preferably sealing cover 2454is provided on the underside thereof with an array of O-rings 2476,shown in FIG. 78C, sealed thereto and arranged so as to sealingly engagea top rim surface of each of sample dishes 2475, when the sealing cover2454 is in place, preferably in removable snap-fit engagement with base2450.

Preferably sealing cover 2454 is provided with an array of diaphragms2477, shown in FIG. 79C, which may be identical to diaphragms 2218described hereinabove with reference to FIGS. 68A-77. Individualdiaphragms 2477 are seated in a ring 2478 mounted over an aperture 2479formed in sealing cover 2454.

Sealing cover 2454 is preferably provided with an array of light guides2480 arranged to receive light from a sample in sample dish 2475 duringlight detection. Light guides 2480 are inserted in apertures 2482 formedin sealing cover 2454. Individual light guides 2480 are provided tocollect light from a sample in sample dish 2475 during SEM inspection.

FIG. 79B shows the apparatus of FIG. 79A with one sample dish 2475positioned at a dish support location 2456 in base 2450. FIG. 79C showssealing cover 2454 in snap fit engagement with base 2450, therebyproviding individual sealing of each of sample dishes 2475 by means ofO-ring 2476.

Reference is now made to FIGS. 80A and 80B, which are simplifiedillustrations of a microscopy multi-sample holder defining a pluralityof SEM compatible sample containers in accordance with a preferredembodiment of the present invention. As seen in FIG. 80A, the microscopymulti-sample holder preferably comprises a base 2500 and a sealing cover2504. The base 2500 is preferably injection molded of a plastic materialand defines an array of sample containers 2506. Each sample container2506 preferably includes an aperture 2508 through which SEM microscopymay take place. An electron beam permeable, fluid impermeable, membrane2510, shown in FIG. 80B, is sealed over each aperture 2508. Membrane2510 is preferably identical to membrane 2110 described hereinabove withreference to FIGS. 58A-67. Sealing cover 2504 preferably is arranged forindividual sealing engagement with each of sample containers 2506.

Sealing cover 2504 is preferably provided with an array of light guides2514 arranged to receive light from a sample in sample containers 2506during light detection. Individual light guides 2514 are inserted intoapertures 2516 formed in sealingcover 2504.

FIG. 80B shows the apparatus of FIG. 80A in sealed engagement, therebyproviding individual sealing of each of sample containers 2506.

Reference is now made to FIGS. 81A and 81B, which are simplifiedillustrations of a microscopy multi-sample holder defining a pluralityof SEM compatible sample containers in accordance with a preferredembodiment of the present invention. As seen in FIG. 81A, the microscopymulti-sample holder preferably comprises a base 2550 and a sealing cover2554. The base 2550 is preferably injection molded of a plastic materialand defines an array of sample containers 2556. Each sample container2556 preferably includes an aperture 2558 through which SEM microscopymay take place.

An electron beam permeable, fluid impermeable, membrane 2560, shown inFIG. 81B, is sealed over each aperture 2558. Membrane 2560 is preferablyidentical to membrane 2210 described hereinabove with reference to FIGS.68A-77.

Preferably sealing cover 2554 is provided with an array of diaphragms2561, shown in FIG. 81B, which may be identical to diaphragms 2218described hereinabove with reference to FIGS. 68A-77. Individualdiaphragms 2561 are seated in a ring 2562 mounted over an aperture 2563formed in sealing cover 2554.

Sealing cover 2554 is preferably provided with an array of light guides2564 arranged to receive from a sample in sample containers 2556 duringlight detection. Individual light guides 2564 are inserted in apertures2566 formed in sealing cover 2554.

FIG. 81B shows the apparatus of FIG. 81A in sealed engagement, therebyproviding individual sealing of each of sample containers 2556.

Reference is now made to FIG. 82, which is a simplified illustration ofa SEM based sample inspection system and a light detection systemconstructed and operative in accordance with a preferred embodiment ofthe present invention. As seen in FIG. 82, a plurality of pre-microscopymulti-sample holders 2600, each containing a multiplicity of SEMcompatible sample containers 2602 of the type shown in FIGS. 58A-77, isshown in an incubator 2604. Preferably, light microscopy inspection ofthe samples in containers 2602 is carried out while the containers 2602are mounted in holder 2600, as indicated at reference numeral 2606, inorder to identify samples of interest Preferably an inverted lightmicroscope 2608 is employed for this purpose.

Preferably automated positioning systems, such as robotic arms, asshown, are used for conveying the pre-microscopy multi-sample holders2600 and the containers 2602 throughout the system, it being appreciatedthat manual intervention may be employed at one or more stages asappropriate.

Thereafter, individual containers 2602 are removed from holders 2600 andplaced on a removable electron microscope specimen stage 2610, which issubsequently introduced into a scanning electron microscope 2612.

The resulting image from the SEM inspection may be inspected visually byan operator and/or analyzed by conventional image analysisfunctionality, typically embodied in a computer 2614.

Preferably, light inspection of the samples in containers 2602 iscarried out while the containers 2602 are in the SEM 2612. Preferably alight guide 2620 is employed to receive light from a light guide (notshown) inserted in container 2602 and to transmit the light to a lightdetector, such as a PMT 2622.

The resulting image from the light inspection may be inspected visuallyby an operator and/or analyzed by conventional image analysisfunctionality, typically embodied in a computer 2624.

Reference is now made to FIG. 83, which is a simplified illustration ofa SEM based sample inspection system and a light detection systemconstructed and operative in accordance with another preferredembodiment of the present invention. As seen in FIG. 83, a plurality ofmicroscopy multi-sample holders 2650, each containing a multiplicity ofSEM compatible sample dishes 2652 of either of the types shown in FIGS.78A-79C, is shown in an incubator 2654. Preferably, light microscopyinspection of the samples in sample dishes 2652 is carried out while thesample dishes are mounted in holder 2650, as indicated at referencenumeral 2656, in order to identify samples of interest. Preferably aninverted light microscope 2658 is employed for this purpose.

Preferably automated positioning systems, such as robotic arms, asshown, are used for conveying the microscopy multi-sample holders 2650containing sample dishes 2652 throughout the system, it beingappreciated that manual intervention may be employed at one or morestages as appropriate.

Thereafter, holders 2650 are placed on an electron microscope specimenstage 2660, which is subsequently introduced into a scanning electronmicroscope 2662.

The resulting images may be inspected visually by an operator and/oranalyzed by conventional image analysis functionality, typicallyembodied in a computer 2664.

Preferably, light inspection of the samples in sample dishes 2652 iscarried out while the sample dishes 2652 are in the SEM 2662. Preferablya light guide 2666 is employed to receive light from a light guide (notshown) inserted in sample dishes 2652 and to transmit the light to alight detector, such as a PMT 2668.

The resulting image from the light inspection may be inspected visuallyby an operator and/or analyzed by conventional image analysisfunctionality, typically embodied in a computer 2669.

Reference is now made to FIG. 84, which is a simplified illustration ofa SEM based sample inspection system and a light detection systemconstructed and operative in accordance with yet another preferredembodiment of the present invention. As seen in FIG. 84, a plurality ofmicroscopy multi-sample holders 2670, each defining a multiplicity ofSEM compatible sample containers 2672, as shown in any of FIGS. 80A-81B,is seen in an incubator 2674. Preferably, light microscopy inspection ofthe samples in sample containers 2672 is carried out holder-wise, asindicated at reference numeral 2676, preferably in order to identifysamples of interest. Preferably an inverted light microscope 2678 isemployed for this purpose.

Preferably automated positioning systems, such as robotic arms, asshown, are used for conveying the microscopy multi-sample holders 2670throughout the system, it being appreciated that manual intervention maybe employed at one or more stages as appropriate.

Thereafter, holders 2670 are placed on an electron microscope specimenstage 2680, which is subsequently introduced into a scanning electronmicroscope 2682. The resulting images may be inspected visually by anoperator and/or analyzed by conventional image analysis functionality,typically embodied in a computer 2684.

Preferably, light inspection of the samples in containers 2672 iscarried out while the containers 2672 are in the SEM 2682. Preferably alight guide 2686 is employed to receive light from a light guide (notshown) inserted in containers 2672 and to transmit the light to a lightdetector, such as a PMT 2688.

The resulting image from the light inspection may be inspected visuallyby an operator and/or analyzed by conventional image analysisfunctionality, typically embodied in a computer 2689.

Reference is made to FIG. 85, which is a simplified partially pictorialand partially sectional illustration of SEM inspection of a sampleconstructed and operative in accordance with another preferredembodiment of the present invention As seen in FIG. 85, a samplecontainer 3000 containing a sample 3002 is seated in a generally centralaperture 3004 formed in a roof 3006 of an electron gun assembly 3010 andis positioned and sized so as to allow impingement of a focused electronbeam 3012 on sample 3002 during SEM inspection.

Container 3000 is seated over an O-ring 3014 located in an interiorsurface 3016 of roof 3006. Container 3000 includes anelectron-permeable, fluid impermeable, membrane 3020 adhered to theunderside of a peripheral ring 3022 of sample container 3000. Sample3002 lies over electron beam permeable, fluid impermeable membrane 3020.

Electron gun assembly 3010, which is part of a SEM inspection assembly,is provided with an electron gun (not shown) operative to provideelectron beam 3012 emitted through a pole piece 3024 in a generallyupward direction.

The electron beam 3012, generated by the electron gun, is shown totravel along a path 3026. The electron beam 3012 passes through electronbeam permeable, fluid impermeable membrane 3020 and impinges on sample3002 within sample container 3000. Electrons backscatter from sample3002 and pass back through electron beam permeable, fluid impermeablemembrane 3020 and are preferably detected by a backscattered electrondetector 3030 in the electron gun assembly 3010.

It is a particular feature of the present invention that the electronbeam 3020 impinges on sample 3002 on the underside thereof therebyinspecting a sample region lying against electron beam permeable, fluidimpermeable membrane 3020.

The electron gun assembly 3010 defines an electron gun assembly internalvolume 3032. Internal volume 3032 is sealed by walls of the electron gunassembly 3010 and the container 3000 so as to maintain an evacuatedenvironment within internal volume 3032, typically at a vacuum of10⁻²-10⁻⁶ millibars, during SEM inspection.

Electron gun assembly 3010 also preferably includes a safety valvesystem comprising an airlock 3040 and a top wall 3042 defining a safetyvalve system internal volume 3044. Prior to removal of container 3000the airlock 3040 is locked, so as to maintain an evacuated environmentwithin internal volume 3044, typically at a vacuum of 10⁻²-10⁻⁶millibars, during container removal. After the airlock 3040 is locked, agas, typically nitrogen, is introduced via inlet tube 3046 into internalvolume 3032 of electron gun assembly 3010. Container 3000 is thenremoved and preferably replaced. Alternatively, another container 3000may then be placed in sample dish assembly 3010. After container 3000 oranother container is introduced into the electron gun assembly 3010, thegas is pumped out) typically through outlet tube 3048, by a pump (notshown) and airlock 3040 is unlocked.

Airlock 3040 also preferably is operative to rapidly isolate internalvolume 3044 from fluid that may enter the electron gun assembly 3010from the ambient, due to leakage through the sample container 3000 oraperture 3004, or through lesions in electron beam permeable, fluidimpermeable membrane 3020.

In accordance with another preferred embodiment of the presentinvention, electron gun assembly 3010 includes an adjustable seat forsample container 3000. The adjustable seat allows high-magnificationimaging of different regions within sample 3002.

Reference is made to FIG. 86A-87B, which are simplified partiallypictorial and partially sectional illustrations of a tissue sampleslicing assembly constructed and operative in accordance with apreferred embodiment of the present invention. As seen in FIG. 86A, aplurality of stacked removable rectangular shaped slabs 3050 are mountedon a stage 3052. Preferably, protrusions 3054 are provided to retainslabs 3050 on stage 3052. A generally central aperture 3056 is formed ineach individual slab 3050 and defines part of a recess 3058. Aconventional slicing instrument 3060, such as a razor blade 3062, ispreferably provided and is operative to slice a tissue sample 3064seated in recess 3058. The tissue sample 3064 is shown in FIG. 86A toextend beyond the top of the stack of slabs 3050.

FIG. 86B shows a plurality of slabs 3050, including a top slab 3066,providing a tissue sample thickness of X1. The slicing instrument 3060slices the tissue sample 3064 into two portions, top portion 3068 andbottom portion 3070, where resulting bottom portion 3070 has a thicknessof X1.

FIG. 87A illustrates the tissue sample slicing assembly of FIG. 86A witha plurality of slabs 3070, providing a tissue sample thickness of X2,where X2<X1 of FIGS. 86A and 86B. As seen in FIG. 87B, the slicinginstrument 3060 slices the tissue sample 3064 into two portions, topportion 3074 and bottom portion 3076, where resulting bottom portion3076 has a thickness of X2.

The present invention provides methods for imaging a wet sample in ascanning electron microscope, while maintaining the sample in a wetstate, at near-atmospheric pressure and at any desired temperature,preferably temperatures in the range of 0-50° C. This is achieved byplacing the sample in a SEM compatible sample container that seals thesample from the evacuated environment of the SEM. The sample is disposedin the SEM compatible sample container so that the sample, or a portionthereof, is positioned in close contact, preferably at a distance ofless than 5 microns, with an electron-permeable partition membrane.Inspection of the sample in a SEM is then performed by placing the SEMcompatible sample container containing the sample in a SEM, anddirecting a scanning electron beam at the sample through the electronpermeable partition membrane.

In accordance with a preferred embodiment of the present invention, SEMinspection methods are provided for a wide variety of biologicalmaterials, such as cultured cells, tissue slices or fragments, organslices, biopsied material, cells in a liquid suspension, fine needleaspirates and samples obtained by lavage of the respiratory system orthe digestive system. Additionally, biological fluids, such as urine,cerebrospinal fluid, milk, saliva, blood plasma, tears, pus, sputum,mucous, interstitial fluid and semen, may also be inspected using themethods of the present invention. Also included in the present inventionare SEM inspection of biological slurries, such as feces and vomit, soilsamples, plant cells, plant tissue, and microorganisms, such asbacteria, fungi, algae, mycoplasma, and viruses. It is appreciated thatthe materials listed above are only representative of the biologicalmaterials that may be inspected using the method of the presentinvention. The materials to be inspected may be homogeneous samples oroptionally heterogeneous mixtures of several different kinds ofbiological materials. Optionally, non-biological materials may also beincluded in the sample.

In accordance with another preferred embodiment of the presentinvention, cells are maintained under cell culture conditions, such asimmersion in growth medium, for example, LB medium at 37° C. forbacteria or in Dulbecco's modified essential medium (DMEM) supplementedwith 10% fetal bovine serum, at 37° C. in a humidified atmosphereincluding 5% CO₂, for cultured animal cells, in a SEM compatible samplecontainer, or in a subassembly thereof, prior to imaging. In thisembodiment, the SEM compatible sample container or subassembly serves asan experimental vessel, analogous to conventional vessels, such as petridishes, cell culture dishes, cell culture flasks, and/or multiwellplates, for growth and/or manipulation of cells. Additionally, samplesincluding cells and other samples may be subjected to varioustreatments, including, but not limited to, addition of mitogens, drugs,hormones, cytokines, antibiotics, toxic materials, viruses, bacteria,vital stains or other staining solutions, mixing (co-culture) ofdifferent cell types, transfection of cells with DNA, irradiation withultraviolet, X-ray or gamma radiation, replacement of the growth mediumwith other media, such as media lacking serum, while in the samplecontainer or subassembly, in accordance with the objectives of anexperiment or an analysis.

In accordance with yet another preferred embodiment of the presentinvention, methods for SEM visualization of non-biological samples andsamples not directly derived from biological material, such as cosmeticcreams, clays, used machine or motor oils, other emulsions andsuspensions, processed food products, and pharmaceutical formulations,are provided.

In accordance with another preferred embodiment of the present inventionmethods are provided for imaging of samples without fixation. Thesesamples include, but are not limited to, animal or human cells culturedin a SEM compatible sample container, animal, human, protozoan, fungal,plant or bacterial cells introduced into the sample container, segmentsof animal, human or plant tissue, or other liquid samples introducedinto the sample container. Fixation processes are employed in microscopyto preserve structural integrity of a sample or of features within asample during the time that elapses until observation and duringpreparative steps, such as permeabilization, dehydration, embedding,thin sectioning, and staining, between sample acquisition and imaging.The methods of the present invention allow imaging of samples withoutperforming any of the aforementioned preparative steps.

In accordance with another preferred embodiment of the presentinvention, samples may be prepared using conventional fixation methods,such as addition of aldehydes, such as formaldehyde, glutaraldehyde orcombinations thereof addition of methanol or addition of osmiumtetroxide, prior to imaging.

In accordance with another preferred embodiment of the presentinvention, methods are provided for visualizing samples without heavymetal stains characteristic of electron microscopy. The efficiency ofelectron backscattering is a function of the material composition of thesample being scanned, varying, for example, with the atomic number or Zvalue of the different atoms that comprise the sample or its components.These quantitative differences in electron backscattering, stemming, forexample, from differences in the Z value of different atoms, providecontrast even in the absence of heavy metal stains. Since the methods ofthe present invention disturb the molecular composition of a sample onlynominally, if at all, components in a sample can be distinguished on thebasis of differences in local concentrations of compounds that differfrom one another in the average Z value. For example, subcellularorganelles in a biological sample can be distinguished based ondifferences in their respective compositions and concentrations oflipids, phosphates, proteins, and salts. Similarly, the components of acomplex mixture, such as those of milk or of sludge, may also be,distinguished on the basis of differences in their material composition,such as in their Z values.

Additionally, samples may be stained with conventional non-specificand/or specific stains. Non-specific stains, such as uranyl acetate,osmium tetroxide, potassium ferricyanide, lead citrate, andphosphotungstic acid, typically comprising a high Z value material,adhere to or otherwise associate with the features of a sample, therebyenhancing the contrast of features of the sample. Conventional specificstains bind or otherwise adhere selectively to specific targetedmolecule or structures, on the surface of the sample or internally, ifthe sample is rendered permeable to the stain by conventional methods.The specific stain may comprise a molecule which has a high selectiveaffinity to a targeted molecule or structure and thereby binds to, orotherwise associates with, the targeted molecule or structure. Examplesof such targeting molecules are antibodies, receptor ligands, hormones,enzymes, enzyme substrates, avidin, streptavidin, lectins, and nucleicacids. At least one high Z-value, EM contrast providing atom may belinked by conventional methods to the high selective affinity molecule.Upon binding to or otherwise associating with the target molecule orstructure, the target molecule or structure is identified when imaged ina scanning electron microscope by the presence of the at least one highZ-value atom linked to the high selective affinity molecule. This methodis often referred to in the art as direct labeling.

Additionally, the at least one high Z-value, EM contrast providing atomneed not be directly linked to the high selective affinity molecule, butmay be attached through indirect labeling methods. The at least one EMcontrast providing atom may be linked instead by conventional methods toa second high selective affinity molecule whose affinity is directedtoward the first high selective affinity molecule. The first highselective affinity molecule is allowed to bind to, or otherwiseassociate with, the targeted molecule or structure and then the secondhigh selective affinity molecule with the at least one linked EMcontrast providing atom is allowed to bind to the first high selectiveaffinity molecule, thereby identifying the target molecule or structure.

A conventional example of such first and second high selective affinitymolecules are two different antibodies from two different organisms,wherein the first antibody from one organism is allowed to bind to thetargeted molecule or structure, and then the second antibody, linked toat least one EM contrast providing atom, from the second organism isallowed to bind to the first antibody bound to the targeted molecule orstructure. This method is known as indirect labeling. The majoradvantage of indirect labeling in the context of the present inventionis that second high selective affinity reagent linked to at least one EMcontrast providing agent are commercially available, eliminating theneed to create de novo such linked molecules.

Another conventional example is where at least one molecule of biotin islinked by conventional methods to an antibody or other molecule with ahigh selective affinity to a targeted molecule or structure, and atleast one EM contrast providing atom is linked either to the proteinavidin or to the protein streptavidin. The biotin-linked antibody isallowed to bind to a targeted molecule or structure, and then either theprotein avidin or to the protein streptavidin, linked to at least one EMcontrast providing atom, is then allowed to bind to the biotin-linkedantibody, thereby identifying the target molecule or structure bound bythe biotin-linked molecule.

An additional conventional example is where a first antibody is allowedto bind to its target molecule or structure, and then either protein Aor protein G, linked to at least one EM contrast providing atom, isallowed to bind specifically to the first antibody, thereby identifyingthe target molecule or structure.

In accordance with yet another preferred embodiment of the presentinvention a method for imaging of thick, “solid” samples, as opposed tocell cultures, cell suspensions, and liquid samples, is provided. Inreference to this embodiment, the term “solid” denotes a range ofconsistencies including hard collagenous matrices, bone, and hard planttissues, as well as soft tissues, such as thymus or pancreatic tissues,and gel-like materials, such as blood clots, agar gels and the like.These samples may include pieces of explanted tissues or tissuebiopsies, natural and artificial gels and. matrices, semisolid media,such as agarose, multicellular assemblies, or thin samples deposited onthe surface of a thick, solid matrix.

In this embodiment, the method includes additional steps to provideclose contact with the partition membrane. To maintain suitable contactthe partition membrane, a sample is provided with a fairly smoothsurface. This surface may be the “natural” edge of the sample, such asan epithelial lining that is part of a tissue sample, or may begenerated by cutting a tissue piece with a razor or a slicinginstrument, preferably by the device and methods depicted in FIGS.86A-87B, or in another preferred embodiment, with a mechanizedinstrument, such as a vibratome®. Additionally, in this embodiment, thetissue typically needs to be pushed against the partition membrane, suchas by a positioning device, preferably, the device shown in FIGS.31A-50.

The sample preparation and imaging are otherwise similar to those forcells, except as described hereinbelow. Tissues may be imaged withoutany treatment, following fixation, or following staining/labeling.Fixation, when used, is either performed by vascular perfusion withfixative or by immersing small tissue fragments in fixative. Stainingand labeling are done using similar procedures to those describedhereinbelow for cells. One important difference is that, whereas samplescomprising cells adhered to the partition membrane of the samplecontainer can be treated by exchanging the fluid in the container,samples comprising tissue fragments are preferably treated beforeintroduction into the sample container. It will be appreciated by thoseskilled in the art that these procedures may have to be modifiedslightly for use with tissue fragments, in view of the empiricalresults.

In accordance with yet another preferred embodiment of the presentinvention, methods are also provided for visualizing a sample and/orspecific structures on the sample. In addition to the detection ofbackscattered electrons, a SEM may be equipped with a photon detector,as shown in FIGS. 58A-84, to detect light produced as a result of theelectron beam scanning. Two significant advantages are associated withthe orientation of the photon detector disclosed in FIGS. 58A-84, whichis different from the conventional orientation. First, the sample iscompletely isolated from the vacuum in the scanning chamber of the SEM,and can be maintained in a wet state at near-atmospheric pressure.Second, the light guide leading to the light detector is generallyplaced at an opposite side of the sample relative to the detectors ofbackscattered electrons, unlike conventional cathodolummescencedetectors, that are placed on the same general side of the sample. Inthe present invention the detectors used for the two modes of detectiondo not compete for the same space, allowing highly efficient and,preferably, concurrent collection of both light and backscatteredelectrons. The scanning electron beam excites, directly or indirectly,molecules in the sample, which then may emit visible light atcharacteristic wavelengths, a process variously known ascathodoluminescence or scintillation. The intensity and/or the spectralproperties of the emitted light can then be used to construct an imageof sample derived from the endogenous scintillating molecules in thesample. Just as inherent differences in the Z values of the differentcomponents or molecules that comprise the sample can be exploited toconstruct a micrograph on the basis of backscattered electrons, theinherent differences in the intensity and wavelength of thecathodoluminescence emitted by the different components or moleculesthat comprise the sample may also be used to construct a micrograph ofthe sample, by conventional methods.

Alternatively, exogenous cathodoluminescence labels known in the art canbe contacted with the sample as a cathodoluminescence stain.Non-limiting examples of such cathodoluminescence labels areFLUOSPHERES, such as Catalog number F-8814, Molecular Probes, Eugene,Oreg., USA, and scintillation proximity assay (SPA) beads, such asCatalog no. RPNQ0006, Amersham Biosciences, Piscataway, N.J., USA.Preferably, the methods of the present invention also provide forspecific cathodoluminescence stains. The specific cathodoluminescencestains are similar to the direct or indirect labeling techniquesdescribed above, wherein instead of the at least one high Z-value, EMcontrast providing atom, at least one cathodoluminescence moiety islinked to the direct or indirect targeting molecules by methods known inthe art A SEM image may be derived on the basis of cathodoluminescencealone or in conjunction with a SEM image constructed on the basis of thebackscattered electrons. In another embodiment of the present invention,the cathodoluminescence detection system is equipped with conventionalmeans for spectral resolution of the light emitted from the scannedsample. Non-limiting examples of such means for spectral resolutioninclude filters, diffraction gratings, and prisms. Thus, this embodimentprovides for measurement of light of a selected wavelength or of awavelength within a selected range, enabling for example, to distinguishbetween light emitted by different materials in the sample, detecting aparticular material or class of materials, or improving the signal tonoise characteristics of the signal.

In accordance with still another preferred embodiment of the presentinvention, further information from the sample may be obtained bydetection and spectroscopic analysis of X-rays emitted from the sampleas a result of interaction with the electron beam, according toconventional methods. Such analysis provides specific and quantitativeinformation on the elemental composition and distribution of the sampleor of regions within the sample.

Additionally, for any given sample, different staining and labelingmethods may be combined as dictated by the imaging and analytic needs.As non-limiting examples, backscattered electrons andcathodoluminescence can be detected during scanning of the same sample,yielding two images of the same region of a sample; in another example,backscattered electron detection can be combined with X-ray detectionfrom either the whole scanned region or from a smaller region, usingconventional methods.

In accordance with another preferred embodiment of the presentinvention, methods are provided for SEM imaging several microns belowthe surface of a sample In conventional SEM the surface topology of asample is imaged using a reflective mode, constructing an image from thedetected secondary electrons dislodged from the surface of the sample bythe scanning electron beam The present invention uses backscatteredelectron detection as the primary detection mode, and such backscatteredelectrons result from interaction of the scanning electron beam withcomponents of the sample that can lay up to a few microns below thesurface of the sample. Since the image is derived from the detectedbackscattered electrons, the actual thickness of the sample is in asense arbitrary, limited only by the depth of the sample container ofthe invention. The contrast of the features of interest, both those onthe surface of the sample and those below the surface, may be enhancedby employing any one or an appropriate combination of non-specific orspecific stains.

In accordance with still another preferred embodiment of the presentinvention, methods are provided for a virtual serial sectioning of asample. The sample is initially scanned with an electron beam at a lowacceleration energy, producing an image of a region of the sample thatis nearest to a partition membrane. Increasing the acceleration energyof the electron beam allows the beam to penetrate into the sample to aslightly greater depth and to derive an image of the interior of thesample at that depth from the resultant backscattered electrons.Subsequent images are obtained in a similar manner by increasing theacceleration energy in stepwise increments, thereby deriving an image ateach level as the electron beam penetrates deeper into the sample, up toa depth of several microns. Acceleration energy values preferably rangebetween approximately 8 keV and 30 keV. The series of images thusobtained serve to identify features lying at different depths within thesample, or may serve as raw data for reconstructing a three dimensionaldistribution of such features, using suitable algorithms.

In accordance with yet another preferred embodiment of the presentinvention, methods suitable for rapid histopathological analysis withinthe time frame of a surgical operation are provided. Surgical pathology,the analyses and results of which are used to determine the continuationand course of surgery, is currently built upon the light microscopicexamination of frozen sections of the dissected tissue. While thistechnique can be accomplished in about 30 minutes, the crude freezingtechniques used cause significant damage to the structure of the tissue,leading to loss of delicate details. The methods of the presentinvention permit a higher resolution SEM examination of the dissectedtissue, within the required timeframe, in the absence of fixation orstaining or drying and under atmospheric pressure, thereby preservingthe delicate detail of the sample. Additionally, non-specific andspecific stains, as well as gentler fixation techniques, may be includedif additional, more detailed, analyses are requiem In accordance withanother preferred embodiment, the methods of the present inventionproviding imaging capabilities may be used to replace or improve anyprocess in research, diagnosis, therapy, industrial or regulatoryinspection, that uses microscopic imaging. As discussed hereinabove,conventional electron microscopy is used to examine a small proportion(3-8%) of specialized cases, such as kidney pathology or oncology, wherethe resolution of light microscopy is insufficient. The methods of thepresent invention permit a more widespread use of SEM for pathology ingeneral and surgical pathology in particular. In addition, the methodsof the present invention may be used instead of, or in addition to,histological or cytological examination that currently use lightmicroscopy. The advantages provided by the methods of the presentinvention may relate to speed, to preservation of the sample in a statemore closely resembling the native state, from obviating the need forthin sectioning, or from other features of the novel imaging mode. Theseadvantages may be particularly important in medical areas in whichcurrent methods are insufficient, such as correct staging of braintumors and of neural pathologies in general, cytological examination ofdispersed cell specimens, such as fine needle aspirations of thyroid orbreast tissue, and blood samples.

The method of the present invention is particularly suitable for use inthe selected applications described hereinbelow. It is appreciated thatthese examples are used for illustrative purposes only, and are in noway intended as limiting.

Obesity and Diabetes: Lipid Droplets in Fat Cells, White and BrownAdipose Tissue

Preparation of samples for conventional TEM involves complete removal ofwater, usually by replacement by organic solvents, such as alcohol orxylene. These solvents dissolve lipids, which are then lost from thesample. Fixation with osmium tetroxide, which cross-links lipidmolecules, works to preserve lipids in thin structures such as lipidbilayer membranes. However, with larger lipid structures, such as lipiddroplets or the lipid component of adipocytes (fat cells), the osmiumreacts quickly with lipids on the surface, creating a “crust” that isless permeable to osmium tetroxide, the interior of the lipid structureis thus left unfixed, and may be destroyed or dissolved later.

The methods of the present invention provide a unique ability to observelipid-rich components in biological samples, by avoiding the need fororganic extraction and lipid fixation. This ability has utility in themedical areas of obesity and diabetes. Treatments for obesity and typeII diabetes effect changes in one or more of the following parameters,which can be directly assessed using the methods of the presentinvention, the distribution of lipids in adipose cells (adipocytes),measurements of number and size of adipocytes, and the distinctionbetween anatomically and metabolically different adipocytes, includingbrown adipose tissue (BAT) and white adipose tissue (WAT), adiposetissue from different anatomical locations, and adipose tissue fromyoung vs. old individuals. Additionally, lipid distribution may bemeasured in adipocytes or preadipocytes derived from humans,experimental animals or in continuous cell lines, as means of diagnosis.

Fatty change in Kidney, Liver

Pathological states of the numerous tissue types, most prominentlyliver, kidney and muscle, are associated with rapid accumulation oflipid droplets inside and outside of cells, termed fatty change. Theclinical observation of fatty change is an early sign of tissue damagedue to alcohol and other hepatotoxins, of various kidney pathologies andof atherosclerosis. More rarely, but significantly, fatty change is alsoindicative of myocardial damage due to infarcts, ischemia, ordegenerative diseases. Monitoring fatty change is also useful during theliver transplantation, to direct decisions on the course of operationand on the suitability of a liver for various patients.

Kidney Pathology

Kidney pathology is one of the major areas for which electron microscopyis presently used for clinical diagnosis. Electron microscopy (EM) isnecessary for the diagnosis of many glomerular diseases, includingminimal change disease, thin basement membrane nephropathy, hereditarynephritis (Alport's syndrome), and fibrillary glomerulonephritis. EMalso permits precise localization of the immune complex deposits, theidentification of tubulo-reticular inclusions in lupus nephritis andHIV-associated renal disease. The methods of the present invention allowrapid and easier performance of high-resolution imaging of kidneysamples.

Toxicology

Toxicological studies are carried out in animals or in humans, thelatter either in ethically controlled studies or in investigating theconsequences of inadvertent exposure. Toxicological effects can often beassessed using microscopic analysis, many toxins affect primarilyselected target organs, most often liver, kidney, lung and the digestivetract. The methods of the present invention provide means for sensitiveand rapid analyses of samples derived from these and other tissues. Suchanalyses have applications, for example, in testing for environmental,occupational and nutritional toxins, as well as for toxic side effectsof drugs. In another preferred embodiment of the present invention, suchanalysis is carried out using X-ray detection, which gives informationon the distribution and concentration of various elements, this hasutility, for example, in the detection of particulate contamination inthe lung or in alveolar macrophages.

CNS: Myelin, MS, Nerve Trauma and Regeneration, and Tumors

The Myelin sheath of nerve processes are prominent lipid-richstructures, which are clearly visible in the methods of the presentinvention. Changes in the extent of nerve myelination and in thestructure of the nerve fibers and the associated myelin sheathsaccompany a wide variety of clinical neurodegenerative situations, suchas autoimmune diseases such as Multiple Sclerosis, Guillain-BarrdSyndrome, congenital storage diseases such as Neuronal CeroidLipofuscinoses (OMIM 256730, possibly the most common group ofneurodegenerative diseases in children), complications of infectiousdiseases such as diphteria, HIV, or prion diseases; and trauma. In thesecases, the methods of the present invention can contribute to diagnosisbased on rapid, high resolution imaging of small samples, such asbiopsies or cerebrospinal fluid, to histopathological analysis in eitherpatients or in animals. Additionally, the ability to observe theorganization of neural tissue at high resolution can be employed in theanalysis of suspected tumors in the nervous system.

Extracellular Matrix

The extracellular matrix (ECM), which is composed mostly of protein,glycoproteins, oligosaccharide and polysaccharide chains, is thestructural foundation of tissues and organs. The ECM plays crucial rolesin diverse processes, including morphogenesis and organogenesis,regulation of cell growth, migration and polar extension, such as axonalgrowth. Several genetic and degenerative diseases are associated withalterations in the structure of the extracellular matrix, such as scurvydue to vitamin C deficiency, which leads to incomplete modification ofcollagen and general dissolution of connective tissues; collagenmodification in aging, resulting in changes in connective tissue;genetic diseases due to mutations in collagen genes, such asOsteogenesis imperfecta (Collagen I mutations), The Helers-DanlosSyntrome, and various arthritic conditions. Additionally, cancer cellsmay have specialized interactions with the surrounding extracellularmatrix, for example, cancer cells produce and secrete enzymes thatchange the structure of the surrounding matrix. Such production andsecretion of enzymes may be associated with specific properties of thecancer cells, including metastatic activity, which may have importantimplications for prognosis and treatment.

The methods of the present invention allow a unique imaging capabilityfor the structure and composition of the extracellular matrix. Suchimaging can provide means of diagnosis, or analysis of drug action inpatients and in experimental animals. In the context of cancer, analysisof extracellular matrix in samples derived directly from tumors, or fromincubation of cancer cells in vitro, can be carried out in accordancewith methods of the present invention; such analysis may provide datafor characterization of the tumor, for designating a treatment protocoland for assessing the progress of the treatment.

Epithelia

The methods of the present invention provide a direct and effectivemeans of viewing epithelia. An epithelial cell layer is placed directlyin a sample container so that it is in contact or in close proximity toa partition membrane, the container is sealed and placed in a SEM, andimaged by scanning electron microscopy according to an embodiment of thepresent invention. The epithelial layer may be imaged while attached toan underlying tissue, or may be removed from such tissues beforeimaging. Additionally,the epithelial layer may occur without suchattachment in its natural position in the body prior to removal forimaging. Applications include the diagnosis of many diseases that affectthe structure of epithelia, such as diseases of the digestive system,the respiratory system, endocrine and exocrine glands, vasculardiseases, skin diseases, eye diseases.

Hematology and Immunology

The methods of the present invention provide means of analysis of cellsof the hematopoietic system, both in the context of a tissue, such as ina biopsy of thymus, lymph nodes or bone marrow, within capillaries in atissue biopsy, or outside blood vessels, especially in sites ofinflammation, or in fluid samples, such as blood or fine-needleaspirates. Information on the abundance or the arrangement ofhematopoietic cells in tissues can be used to derive diagnosis and todirect decisions on treatment protocols in a variety of pathologicalsituations, including inflammatory diseases, such as rheumatoidarthritis, lupus, other autoimmune diseases, atherosclerosis, and woundhealing. Another application of unique importance is the early andaccurate detection of rejection of transplanted tissues, such as aheart. Recipients of such transplanted tissues are generally placedunder immuno-suppressive treatment for extended periods to preventrejection of the transplant. However, the transplant must be frequentlymonitored for early signs of rejection, which must be met with immediateadjustment of treatment Such monitoring is currently achieved bymicroscopic examination of tissue biopsies taken, for example, at weeklyintervals. The methods of the present invention may provide a moresensitive or accurate means of assessing the state of the transplantfrom such biopsies.

Cancer

Microscopic imaging of tumors, biopsies, needle aspirations, or cellsuspensions from suspected tumors, of portions of surgically removedtumors, and of cells derived from tumors and maintained in vitro have animportant role in diagnosis, typing, and grading of tumors, as well asin determining the prognosis and course of patient treatment. Themethods provided in the present invention provide new capabilities formicroscopic imaging, such as high resolution, easy and rapid samplepreparation, reduction of artifacts due to sample preparation, andunique contrast mechanisms, which may be used for improvements or asunique tools in diagnosis, typing, and grading of tumors, as well as indetermining the prognosis and course of patient treatment

Microbiological Entities

In accordance with another preferred embodiment of the presentinvention, the method provides for imaging of microbiological entities,including, but not limited to, bacteria, archea, fungi, protozoa,Giardia, Pneumocystis carinii, bacterial and fungal colonies, mycelia,bacterial and fungal biofilms, and viruses. Microbiological entities maybe imaged in several types of samples, such as blood, sweat, tears,stool, sputum, stool, urine, pus, cerebrospinal fluid, tissue specimens,lavages of the lungs; bronchia, or digestive system, environmentalsamples, soil samples, and plant samples. Such inspection may be of usefor detecting microbiological contamination in patients, animals orplants, or on medical, industrial or household devices.

Additionally, SEM inspection of microbiological entities may be used fordetection of the presence of such entities in a sample, foridentification of the entities, as for example distinguishing bacteriafrom fungi or viruses, or determining a broad or unique classificationof a microbiological entity. Additionally, SEM inspection may be used tocharacterize some property of a microbiological entity. For example, thesensitivity or resistance of a bacterium to one or more antibioticsubstances may be rapidly examined by incubating small numbers of suchbacteria with the antibiotics, preferably using different concentrationsof antibody in each of several parallel experiments, and after a periodsufficient to detect an effect on individual cells, typically less thanone hour for penicillin, inspecting the bacteria by SEM according to themethods of the present invention. Antibiotics may induce morphologicalchanges or lysis of susceptible bacteria, while the same antibiotic maynot induce such changes in resistant bacteria, thus establishing acriterion for rapid assessment of antibiotic susceptibility orresistance in the bacteria.

Cell-Coated Devices, Implants, Stents

Another aspect of the present invention provides for imaging of samplesderived from devices or formulations used as biomedical implants or ascell carriers for biotechnology, or other medical or industrial devicesor formulations that may come in contact with biological materialsduring operation or storage. A common feature for these samples is thatcells, including human, animal, protozoa or microbial cells, are adheredor grown on a surface of the device or formulation. The device orformulation may be of such thickness or material composition that isgenerally impermeable to electrons or to light, thus precluding directimaging in a transmission electron microscope or by transmitted lightmicroscopy. Using methods of the present invention, such samples can beplaced in a sample container so than the surface of interest is proximalto the electron-transparent partition membrane. As discussed, thereflective mode of imaging of the present invention allows to image thesurface layer of the sample, including any cells present on the surface.Examples of such samples include cardiovascular implants such as stents,covered stents, valves, bypass or replacement arteries. These devicesare typically made of metal, synthetic or natural polymers or tissues,or combinations thereof. Major concerns with the use of such devicesinclude the presence of cells, especially fungi and microbes, beforeimplantation, the coverage by normal endothelial, and the prevention ofunwanted reactions leading to clot formation. Implants may be examinedbefore implantation, in experimental or quality control conditions,after incubation with cells or bacteria in vitro, after implantation inanimals, or after implantation in humans, conceivably after the implantis no longer required or after its failure.

Implants such as the cardiovascular implants or, prostheses, sutures,hard or soft tissue implants may additionally be examined by methods ofthe present invention as part of the manufacturing process to deriveinformation on the material and structural properties, or on any otherfunctional aspect of the implants, such as response to a variety oftreatments in vitro or in vivo. The examination may occur duringdevelopment of the implants or of materials and parts used in themanufacture of the implants. In other embodiments, the examination maybe performed on a sample of part of a manufacturing batch or of everymanufactured implant.

Medical devices which may suffer deposition of unwanted materials onsurfaces, including various catheters, kidney dialysis devices, infusiontubing, endoscopy devices, and any containers or tubings that maycontact materials that are to be used medically or nutritionally mayalso be examined by methods of the present invention. Examples ofmaterials that may be deposited on the devices include bacteria,bacterial biofilms, fungi, protozoa, mycoplasma, organic or inorganicprecipitates or adhered phases, blood cells such as platelets,macrophages, leukocytes.

Additionally, materials and formulations used in tissue engineering,typically including a synthetic or biologically derived matrix and,optionally, cells deposited within or on the surface of such a matrixmay also be examined by methods of the present invention. Vessels,tubings, filters, substrata for cell attachment or growth includingmicrocarriers, fiber beds, hollow fibers and stacked plate modules, usedin biotechnology and bioengineering applications may also be examined bymethods of the present invention. Non-limiting examples of cell growthmicrocarriers include CYTOLINE® and CYTODEX® from Amersham Bioscienceand CULTISPBER® from Hyclone of Logan, Utah, USA, hollow fibers includeFiberCells® from Fibercell systems of Frederick, Md., USA The vessels,tubings and substrata come in contact with cultured cells, with cellgrowth media, and/or with cleaning solutions. The arrangement, density,integrity and structures of cultured cells and of layers and aggregatesof such cells may have important consequences in the biotechnologicalapplications of such cells, and can be monitored and analyzed usingmethods of the present invention. Deposition or growth of undesiredsubstances and organisms on the vessels, tubings and substrata,including aberrant cells or cell assemblies, microbes, microbialbiofilms, fungi, protozoa, mycoplasma, organic or inorganic precipitatesor adhered phases, can also be monitored and analyzed using the methodsof the current invention.

Quantitative Pattern Analysis of Biomolecules

In another aspect of the present invention, methods are provided fordetermining the quantity and spatial distribution of specific moleculesin a sample. The need for such analysis arises in a multitude ofsituations, some of which are detailed hereinbelow.

Quantitation of biomolecules in biological samples is currently done bya variety of means, including:

-   -   1. Biochemical and immunological assays in which the total        amount of a biomolecule in multiple cells is measured; for        example, radioligand binding assays, radioimmunoassay,        fluorescence-based binding assays, assays of enzyme activity,        immunoblotting, etc. While yielding accurate measures of the        total quantity of biomolecules in the sample, such methods do        not give any information on the spatial distribution, on        differential concentration in different cells in the sample,        etc. Furthermore, two issues of sensitivity arise: first, limits        on sensitivity of detection may necessitate the use of a large        number of cells or a large amount of tissue for measurement.        Second, biomolecules that are highly concentrated in a limited        region of the cells, such as a subcellular organelle, or of the        tissue sample are diluted with an excess of material from        regions of the sample that do not contain the biomolecules, thus        limiting the sensitivity of detection.    -   2. Antibody, ligand or enzyme-based staining of cytological or        histological samples, and detection by light microscopy.        Typically, a sample is incubated with a solution containing        antibodies that bind specifically to a biomolecule of interest.        The antibodies are linked, either directly or indirectly, to a        labeling moiety that is visible by light microscopy. Such        labeling moieties may include fluorophores, chromophores,        particles that are either opaque to light or scatter light, or        enzymes that generate localized concentration of fluorophores,        chromophores, opaque or highly scattering materials. Such        methods, coupled to imaging and image analysis with light        microscopy, yield information on both the amount and        distribution of the target biomolecules. Limitations of such        techniques include resolution (limited by light diffraction to        approximately 250 nm), signal to noise ratio, especially when        analyzing biomolecules with a diffuse distribution, sensitivity,        bleaching of fluorescent dyes, limited dynamic range and        non-linear response.    -   3. Antibody or ligand-based staining of cytological or        histological samples, and detection by electron microscopy (EM).        Similar in general principle to light-microscopy based methods,        the detection here is based on electron-dense or        electron-scattering substances, including for example colloidal        gold particles, ferritin, precipitates such as lead phosphate,        and polymerized diaminobenzidine stained with heavy metals or        osmium tetroxide. EM-based detection, quantitation and pattern        analysis of biomolecules can reach very high resolutions (better        than 1 nm in transmission EM, 5-10 nm in scanning Ed. A        comparative analysis of EM based relative to fluorescence based        quantitation of biomolecules (Levit-Binnun N, Lindner AB, Zik O,        Eshhar Z, Moses E., 2003. Quantitative detection of protein        arrays. Anal. Chem. 75(6):143641; and patent application        WO02/14830-PCT/IL01/00764) documents a significant advantage of        EM-based detection of gold colloids in sensitivity, signal to        noise ratio, and dynamic range.

Current methods for EM-based quantitation and pattern analysis ofbiomolecules in biological samples such as cells or tissues requiresample preparation procedures typical of electron microscopy. Fortransmission EM, these require embedding or rapid freezing followed byultrathin sectioning. One resulting limitation is that only a small, andoften arbitrary, portion of the cell or tissue is present in the thinsection. For example, when attempting to detect and quantify proteinsassociated with the cell membrane, a thin section will include only avery small portion of the entire cell membrane. Labeling and imagingintact or permeabilized cells, is made possible when scanning EM is usedfor detection, where the samples do not need to be thin sectioned forimaging. Standard SEM, in which the sample needs to be dried by methodssuch as critical point drying, suffer from artifacts due to the dryingprocess, as well as problem in clear identification of the labelingparticles when the samples are coated and imaged by secondary electrondetection.

The methods of the present invention provide a mode of detection andquantitation of biomolecules that uniquely overcomes many limitations ofexisting techniques. The EM-based detection provides for highsensitivity, dynamic range, and signal to noise ratio; the highresolution imaging and the optional use of discreet labeling moietiessuch as colloidal gold particles, allow to map with high precision thespatial distribution of biomolecules; the ability to observe intact, orpermeabilized but otherwise intact, cells, yields a global view of thedistribution of biomolecules throughout the cell or the cell surface orthe tissue examined; and the ability to observe cell and tissues withoutdehydration, drying and embedding results in the elimination ofpotential artifacts associated with these processes.

Non-limiting examples of applications in which the quantity and spatialdistribution of biomolecules are important parameters include:

Distribution of receptors on the cell surface. Cell surface receptorsfor signaling molecules such as hormones, cytokines, chemokines, growthfactors, extracellular matrix, cell:cell interactions, cell adhesionmolecules, and cell: pathogen interactions are often distributed on thecell surface in a non-uniform distribution, and both the quantity anddistribution of such receptors may affect the reactivity to, signals andmay be modulated in response to signals. Thus, the quantity anddistribution of receptors on the cell surface in cells and in tissuesare important predictors of the properties, of potential responsivenessto external stimuli, and of the cells and tissues. Additionally, changesin the quantity and distribution of receptors upon application ofsignals may yield unique information on the response to the signals.Extensively studied cases include the dimerization and multimerizationof growth factor receptors such as the Epidermal Growth Factor (EGF)receptor. The EGF receptor, which may be in monomeric form prior tobinding of EGF, rapidly aggregates into dimers and larger multimers uponbinding of EGF. This aggregation is a necessary and sufficient step inat least part of the signal transduction process. After the initialaggregation, receptors may undergo additional processes that change thequantity and surface distribution, namely further aggregation, andinternalization into intracellular compartments not accessible fromoutside the cell. These processes also have important roles in signaltransduction, in modulating the duration of the activated state, and inmodulating the sensitivity to subsequent signals.

Association of biomolecules with identical or distinct biomolecules.Biomolecules such as proteins often function as dimers or largeroligomers, in which the subunits may be identical (homodimers,homo-oligomers) or different (heterodimers and hetero-oligomers). Forexample, the EGF receptor, also designated HBER1, can form dimers withan other HER1 molecule, or with any of a group of similar receptormolecules termed HER2, HER3 and HER4; in fact, the HER1-4 proteins canassociate in a variety of homodimers and heterodimers. The formation ofeach such dimer may be differentially influenced by binding of any ofseveral EGF-like ligands, with different biological consequences.Finally, the HER family of receptors are differentially expressed insome types of cancer; a much-discussed example is the high prevalence ofHER-2 in some breast cancers. Indeed, high prevalence of HER2 in acancer is liked to a poor prognosis, and drugs such as Herceptin® targetthis receptor specifically. The association of receptors and otherproteins into dimers and larger oligomers has been investigated usingbulk techniques, in which (for example) a general crosslinking moleculeis used to covalently link molecules that are in close proximity, or bydistance-dependent energy transfer between fluerent molecules andorophores attached to different proteins. The methods of the presentinvention uniquely allow to measure the extent of association of similaror different biomolecules, by using labeling each of the biomoleculeswith a label that can be distinguished from each other by SEM inspectionaccording to the methods of the present invention. Non-limiting examplesof such distinguishable labels are colloidal gold particles of differentsizes (e.g. 10 nm and 20 nm, 15 nm, 25 nm and 35 nm, etc.), andcombination of electron-dense labels with cathodoluminescence labels,visualized in the same sample by simultaneous detection of backscatteredelectrons and light

Emulsions and Suspensions

Emulsions, comprising a fine mixture of immiscible phases such as oiland water, and suspensions, which are fine mixtures of solid particlesin a liquid, are very difficult to analyze using current electronmicroscopy techniques. Nearly any method for fixation or freezing, maydamage or significantly alter the microscopic structure of the sample.Observation of such samples using environmental SEM is also unreliabledue to the difficulty in maintaining the composition of the unfixedsample.

The methods of the present invention provide a unique means of imagingsuch samples such as dairy products, other food products includingbutter, margarines and substitutes, cosmetic creams, sunscreen creams,machine and motor oils and lubricants, clays, and pharmaceuticalformulations, biological fluids such as milk, blood, plasma and serum,feces, and environmental samples.

Automated SEM

It is another objective of the present invention to provide means forautomated electron microscopy of wet samples, and specifically ofbiological samples. Such automated microscopy has been widely applied inthe semiconductor industry. The main barrier to the application ofautomated electron microscopy to wet samples is the need to employsample preparation procedures such as drying, embedding, sectioning orcoating, which are highly complex and not amenable to automation. Thepresent invention provides means for direct imaging of wet samples in ascanning electron microscope, thus obviating the need for theaforementioned preparative procedures. In fact, the introduction ofbiological or other wet samples into the sample container and thepreparation for imaging involve, at most, a series of additions andremovals of liquids to a dish. Such manipulations, carried out byappropriately designed pipettors and fluid aspirators, are easy toautomate, as is done on a massive scale in applications such as drugscreening and genomic sequencing. Automated sample preparation providedby the methods of the present invention, which may be coupled to otherautomated steps known in the art such as automated introduction into aSEK, automated positioning, registration of location markers, selectionof area of interest for observation, and image analysis, can provide foressentially any level of automation required. A non-limiting example ofthe utility of such automated systems include drug screening usingparameters measured at high resolution and disclosed in the precedingsections, such as drug-induced structural changes in cells ordrug-induced changes in abundance or distribution of cell surfacereceptors; and quality assurance applications, for example in the drugor other chemical industries.

GENERAL PROTOCOLS OF THE PRESENT INVENTION

A general outline of the protocols used in the methods of the presentinvention is disclosed hereinbelow. It will be appreciated by thoseskilled in the art of electron microscopy that these protocols aregeneral in scope and may be adapted or modified to suit thecircumstances of the particular sample being examined and the desiredanalyses.

Reference is now made to FIG. 88, which depicts schematically the mainprotocol steps that comprise the method of the present invention. It isappreciated that not all embodiments will comprise all of the stepsenumerated hereinbelow. It is appreciated that the methods hereindisclosed provide for a desirable degree of flexibility in preparing andvisualizing the sample.

The individual steps comprising the protocol are now explained ingreater detail. In all protocols, unless specified otherwise, alloperations are performed at room temperature; and a “wash” meansremoving the liquid from the sample container, adding the appropriatewashing liquid, then incubating for approximately 5 minutes.

1. Coating the Electron-Transparent Partition Membrane

The sample to be imaged must be held in close proximity to, andpreferably contacting, the electron-transparent partition membrane. Somesamples may adhere directly to the partition membrane without specialtreatment. Other samples adhere better if the membrane is coated with anadhesion agent before sample is placed into the container and applied tothe partition membrane. A non-limiting example of a general adhesionagent is poly-L-lysine. Poly-L-lysine can be used with a variety ofsamples, including non-adherent animal cells (eg. blood cells),bacteria, protozoa, and a-cellular particles (e.g. particulate matter ina suspension). Other non-limiting examples of adhesion agents known inthe art are extracellular matrix components, such as fibronectin,collagen, gelatin, laminin, or Matrigel® (Invitrogen). Extracellularmatrix components are typically used as adhesion agents when cultured orprimary animal cells are to be maintained on the partition membrane forsome duration (several hours or days) under culture conditions. Theparticular adhesion agent is then selected on the basis of the sample'sparticular growth requirements and the nature of the biologicalexperiment performed, if any, on the partition membrane.

The following are non-limiting examples of methods of the invention thatmay be used to coat the electron-transparent partition membrane.

(a) Fibronectin Coating

Materials

Fibronectin 0.1% w/v (e.g. Catalog No. F-1141, Sigma Chemical Co.).

PBS (Phosphate-buffered saline, e.g. catalog No. 14040, Invitrogen)

Procedure

Dilute fibronectin 1:10 with PBS (final concentration of fibronectin,0.01%).

Apply 15 μl of the diluted solution onto the partition membrane andincubate for 30 min at room temperature.

Remove the solution and wash 5 times with PBS.

Wash twice with the appropriate growth medium (e.g alpha-MinimalEssential Medium, α-MEM with 10% fetal bovine serum).

(b) Poly-L-ysine Coating

Materials

Poly-L-lysine 0.1% w/v in water (e.g. cat no. P8920 from Sigma ChemicalCo.) PBS

Procedure

Apply 15 μl of poly-L-lysine 0.1% solution on partition membrane andincubate for 1 hour at room temperature.

Remove the solution and wash 5 times with distilled water.

Wash twice with PBS or appropriate medium.

2. Placing Sample in Container and Applying to Partition Membrane

Liquid samples or cell suspensions are placed in the container andapplied to the partition membrane by pipetting the liquid or cellsuspension directly into the container on to the membrane. Cells andparticles will usually reach the membrane by gravity or by random motionand will adhere. Adhesion may be a passive process, mediated for exampleby electrostatic interactions; in the case of animal cells, adhesion isoften a specific process mediated by receptors which may depend onmetabolic energy, and is often followed by cell spreading on thepartition membrane. If the cell concentration is low or the cells arenot easily adsorbed on to the membrane, the cells may be centrifugedgently in the sample container (e.g., 5 minutes, 500×g), therebyconcentrating the cells onto the partition membrane.

Additionally, cells may be grown on the optionally coated partitionmembrane under conditions identical or very similar to growth onstandard polystyrene tissue culture dishes. The cells will spread,multiply, interact with neighboring cells and respond to exogenous orendogenous signals. Typically, the cells are maintained in growth mediumsuch as α-MEM, Dulbecco's Modified Essential Medium (DMM) or Ham's F-12,supplemented with 10% fetal bovine serum, in a humidified atmospherewith 5% CO₂, at 37° C. In addition, it is possible to transfect thecells with DNA or to infect the cells with viruses or recombinant viralvectors.

As depicted in FIG. 88, the sample may be imaged without any additionalprocessing (see step 7 below), or optionally processed fierier so toenhance the sample's contrast or to label specific components, asdescribed hereinbelow.

3. Fixation

Although it is possible to optionally stain the sample bothnon-specifically and specifically without prior fixation, the sample isusually fixed prior to optional staining. Fixation preserves thecellular structures as close to the living state as possible andprotects the sample from morphological alteration and damage during thesubsequent treatments. Additionally, fixation can permanently stabilize(“freeze“) the sample in a particular state for subsequent observation,and is particularly useful if transient states are to be visualized.

A wide range of fixation methods known in the art for cytological,immunostaining and electron microscopy may be used. No one fixativepreserves all the cellular structures, thus correct choice of fixativewill depend on the sample, and on the particular features to bevisualized. If the sample is to undergo specific staining such asimmunostaining subsequent to fixation, the nature of antigen andantibody will also affect the selection of a fixation procedure.

Fixation typically involves washing off the culture medium, incubatingthe sample with a fixative solution for the appropriate time interval,and then removal of the fixative.

The following are non-limiting examples of fixation protocols that maybe used with the methods of the invention. Optionally other fixationprocedures known in the art may also be used as dictated by theparticulars of the sample, staining, labeling, and visualizationobjectives.

Unless otherwise specified, all methods described henceforth areperformed at room temperature.

(a) Mild Formaldehyde Fixation (Optionally Used Before SpecificStaining, to Minimize the Damage to Targeted Sites):

Materials

Paraformaldehyde, EM grade (for example 16% solution, ElectronMicroscopy Sciences, cat no. 15710)

PBS

Procedure

Prepare paraformaldehyde 4% solution in PBS.

Wash the sample several times with PBS.

Fix with paraformaldehyde 4% at room temperature for 15 min.

Wash five times with PBS.

(b) Glutaraldehyde/Formaldehyde Fixation (Optionally Used Prior toAggressive Staining Procedures, such as Uranyl Acetate).

Materials

PBS

Wash solution: 0.1 M sodium cacodylate, pH 7.4, 1% sucrose, 5 mM CaCl₂.

Fixative solution: 2% glutaraldehyde, 3% formaldehyde in wash solution.

Procedure

Wash the sample 5 times in PBS

Incubate in fixative solution for 1 hour at room temperature

Wash 5 times with wash solution

Wash 5 times with water.

(c) Cold Methanol Fixation (Optionally Used Before Specific Labeling,when Treatment with Formaldehyde may Interfere with Such Labeling)

Materials

Methanol cooled to −20° C.

PBS

Procedure

Wash the sample 5 times with PBS at room temperature.

Place the sample container on ice, taking care to avoid direct contactof the partition membrane with the ice.

Remove the PBS and add pre-cooled (−20° C.) methanol. Transfer to −20°C. for 5 min.

Wash 5 times with PBS at room temperature.

Proceed to staining or labeling reaction.

4. Non-Specific Staining.

The overall electron nicroscopic contrast of the sample may be enhancedby use of non-specific stains and staining procedures known in the art.What is common to these procedures is that they result in the adsorptionor concentration of high atomic number (Z-value) atoms on the sample oron to structures in the sample.

The following are non-limiting examples of staining procedures known inthe art Optionally other staining procedures known in the art may beused as dictated by the sample, labeling, and visualization objectives.

(a) Uranyl Acetate (UA) Staining.

Materials:

Tannic acid, 1% (w/v) solution in water (prepared from e.g. cat. No.Sigma Chemical Co.).

Acidic uranyl acetate (UA) concentrate: 5% (w/v) UA in water, adjustedto pH 3.5 with HCl.

UA staining solution: 0.1% UA, prepared freshly by dilution from 5% UAconcentrate in water.

Procedure:

Fix sample as described in the glutaraldehyde/formaldehyde procedure.

Wash 5 times in distilled water.

Add tannic acid solution, incubate 5 minutes.

Wash 5 times in water

Add UA staining solution, incubate 20 minutes.

Wash 5 times in water. Leave in water for imaging.

Osmium Tetroxide (OsO₄) Staining.

Material:

OsO₄ staining solution: 1% (w/v) osmium tetroxide in water, diluted fromcommercial 4% stock solution.

Procedure:

Fix sample as described in the glutaraldehyde/formaldehyde procedure.

Wash 5 times in distilled water.

Add OsO₄ staining solution, incubate 30 min.

Wash 5 times in water. Leave in water for imaging.

5. Specific Labeling

The location and quantity of particular molecules and/or structures in asample may be measured by the use of specific targeting molecules thatbind or otherwise associate with the targeted molecules and/orstructures. At least one high Z-value atom directly or indirectly linkedto the specific targeting molecule provide the contrast enhancement forthe targeted molecules and/or structures. Alternatively, the specifictargeting molecules may lead to the localized accumulation of substancesthat generate measurable contrast in the vicinity of the targetedmolecules and/or structures when visualized by the methods of thepresent invention.

Non-limiting examples of commercially available molecules that can beused as specific targeting molecules that bind or otherwise associatewith the targeted molecules and/or structures include antibodies,protein A, and streptavidin; antibodies, protein A and streptavidin towhich colloidal gold particles have been linked; and antibodies, proteinA and streptavidin to which horseradish peroxidase has been linked.

It will be appreciated by those skilled in the art, that the labelingmoiety need not be limited to high Z value atoms. If the SEM instrumentis equipped with a photon detector and/or an X-ray detector, thespecific targeting molecules may be linked moieties that emit light orX-rays in response to the electron beam instead of the high Z valueatoms.

Generically, specific labeling procedures consist of four or five mainsteps, depending on whether the at least one high Z value atom isdirectly or indirectly linked to the targeting molecule: optional samplepreparation, optional fixation, optional blocking of non-specificbinding, binding of the targeted molecule or structure by the targetingmolecule, and optional removal of excess, unbound targeting molecules.If the targeting molecule is already linked to at least one high Z-valueatom, then the labeling process is complete and the sample is ready forvisualization. If the targeting molecule is not so labeled, then one ormore added steps are needed prior to visualization in which high Z-valueatoms are bound to the first targeting molecule by a second targetingmolecule.

Surface molecules or structures can be labeled on live or fixed cells.Intracellular antigens can be labeled on fixed, permeabilized cells. Itwill be appreciated by those skilled in the art that fixation procedurescan mask or change some epitopes, and thus optimal fixation procedurefor each targeting molecule should be determined experimentally. It willalso be appreciated by those skilled in the art that, optimal blockingfor non-specific background, concentration of the targeting molecule,and incubation time, are parameters that depend on the targeted moleculeand/or structure and targeting molecule in question. In some casesspecific incubation and wash buffers are required to avoid non-specificbinding. Thus, there is no standard procedure that works for alllabeling reactions. Optimal conditions may be established based on priorexperience with the particular targeting molecules and targets, or byconducting preliminary experiments using fluorescent labels andvisualization in a fluorescence microscope.

The following are non-limiting examples of specific labeling proceduresknown in the art. Optionally other specific labeling procedures known inthe art may be used as dictated by the sample, staining, andvisualization objectives.

(a) Specific Labeling of Cell Surface Receptors

Materials

PBS

Blocking agent: bovine serum albumin, (BSA) or normal serum.

Primary or first antibody targeted to desired receptor

-   -   Gold particle conjugate capable of binding to or otherwise        associating with the primary or first antibody

Distilled H₂O

Silver enhancement kit, (e.g., AURION R-GENT SE-EM® cat no. 500.033)

Procedure

Fix cells with the mild formaldehyde procedure.

Incubate cells with protein containing solution, such as 3% BSA (bovineserum albumin) or, if indirect labeling via a secondary antibody is tobe used, 1-5% normal serum from the species of the secondary antibody,in PBS, for 30 min.

Incubate cells with primary antibody in the same blocking solution asabove (typically 30-60 minutes). For some antibodies labeling can beimproved by incubating at 37° C. or by longer incubation (several hoursto overnight) at 4° C.

Perform control reaction as above but without primary antibody.

Wash cells several times with PBS.

Incubate with the gold labeled conjugate (e.g., gold conjugatedsecondary antibody targeted against primary antibody, or gold conjugatedprotein A or G; if primary antibody is biotinylated, gold-conjugatedstreptavidin or avidin can be used) in protein containing solution, suchas 1-3% BSA or 1-5% normal serum.

Wash extensively with PBS to remove unbound antibodies.

If the colloidal gold particles are too small to be effectively viewedin the SEM (typically less than 30 nm in an SEM with a thermal electrongun, or less than 10 nm in an SEM with a field emission electron gun),silver enhancement can be used to increase the size of the high-Zparticles. Silver enhancement can be done using commercially availablekits such as AURION R-GENT SE-EM® cat no. 500.033.

Wash several times with distilled water.

(b) Specific Labeling of Intracellular Molecules or Structures:

The procedure for specific labeling of internal molecules or structuresis essentially the same as for surface molecules except that the cellsmust be permeablized after fixation.

Additional Materials

0.2% Triton X-100 in PBS

1% Triton X-100 in PBS

Procedure

For paraformaldehyde or glutaraldehyde fixed cells, permeabilize thecells by incubating with 0.2% TritonX-100/PBS for 5-15 min. (Whenmethanol fixation is used, no additional permeabilization is required).

Wash five times with PBS.

Perform specific labelling procedure as described above. If subsequentvisualization reveals problems with excessive background signal, add amild detergent such as Triton X-100/NP40 (1% v/v) to the wash bufferused after incubation with primary antibody.

It is preferable to use small gold particles (e.g. 0.8 nm colloidal goldparticles) or other contrast-generating materials (e.g.peroxidase/diaminobenzidine/osmium tetroxide) that penetrate the fixed,permeabilized cells more readily than large (e.g. 2040 nm) colloidalgold particles.

(c) Specific Labeling Using Enzyme-Linked Targeting Molecules.

In another method of the present invention, specific labeling isachieved without having the high-Z contrast generating agent directlylinked to targeting molecules. Rather, the targeting molecule causes thelocal accumulation of contrast-generating molecules in its vicinity. Asa non-limiting example, this can be achieved by an enzymatic or otherchemical reaction that causes precipitation of a material containinghigh Z number atoms. An example known in the art is a labeling procedurethat uses the enzymatic activity of horseradish peroxidase (HRP) that iscovalently linked to an antibody. The enzyme catalyzes thepolymerization and insolubilization of diaminobenzidine (DAB); thepolymerized DAB binds and accumulates high-Z reagents such as osmiumtetroxide or metallic compounds with a much higher affinity thansurrounding material in the sample, yielding high contrast

The following is a non-limiting example of a specific labeling procedureusing HRP.

Materials:

All reagents for fixation and immunostaining as described above; thesecond antibody is conjugated to HRP (e.g. cat. No. Amersham)

Diaminobenzidine staining kit (e.g. cat. No. SK-4100, VectorLaboratories) 0.05% osmium tetroxide in water (diluted from 4%concentrate)

Procedure

Fix sample with mild formaldehyde procedure, stain with first antibodyas described above.

Incubate with secondary antibody, anti-mouse peroxidase (1:100 dilution,1 h).

Wash 5 times with PBS.

Wash 3 times with distilled water.

Perform DAB staining (Vector laboratories, cat no. SKA-4100) accordingto the kit protocol

Wash with water ×3

Incubate with 0.05% Osmium tetroxide for 1 min.

Wash 5 times with distilled water, retain in water until imaging.

6. Specific Labeling Without Fixation (Limited to ExtracellularMolecules or Structures).

It is often possible to specifically label surface proteins orstructures without fixation using the above procedure, assuming thetargeted surface proteins or structures do not redistribute orinternalize into the cell while the procedure is being carried out.Performing the incubations at 0°-4° C. may inhibit the targetredistribution or internalization processes. Incubation times may thenhave to be increased accordingly.

7. Imaging

After sample preparation is complete, and the sample is disposed withinthe sample container, the sample container is sealed, for example byengaging a bottom and a part of the container, and is placed on a samplestage in an SEM. Imaging is done either at high vacuum or at “lowvacuum” modes, as the degree of vacuum outside the sample container doesnot substantially affect the imaging or the stability of the samples.However, as a precaution, the SEM imaging may be perform at low vacuumconditions to prevent possible contamination of the microscope in therare cases in which the partition membrane is ruptured.

Imaging is done with an electron beam, generally set from 10 to 30 KeV.Imaging parameters (beam energy, current, spot size, scan rate, contrastand brightness settings, etc.) are used that are optimal for the sample,as determined by a skilled SEM operator.

Alternatively, the sample may be fixed, stained and/or specificallylabeled before introduction into the sample container. This option isimplicitly depicted in the leftmost arrow in FIG. 88, whereby a samplethus treated is also considered a “sample”. Such treatment of the sampleprior to introduction into the sample container may be applicable inparticular for tissue samples, for which treatments are difficult toperform after introduction into the sample container, but also forcellular or other liquid samples.

The following protocols exemplifies preparation and immunostaining ofnon-adherent cellular samples prior to introduction into the samplecontainer and onto the partition membrane.

The present invention provides methods for imaging thick solid samples.SEM inspection of such samples according to the methods of the presentinvention minimally entails placing the surface of the sample to beimaged in close contact with an partition membrane of the samplecontainer of the present invention, sealing the sample container,placing the sample container in a scanning electron microscope, andscanning the sample with an electron beam.

Preferably, the imaged surface of the sample should be such that can bemade to contact the partition membrane over a substantial area Inpractice, this means that the imaged surface should be generally flat,or of moderate curvature or irregularity, either before the sample isplaced in the sample container, or after application of pressure to abutthe sample against the partition membrane.

A solid sample may have such a surface, for example a natural edge of atissue or organ, or an epithelial layer that lines a tissue. In othercases, for example when internal regions of a solid tissue are to bevisualized, the image surface is generated by cutting the sample. Thiscutting can be achieved manually with a scalpel or razor blade; usingrazor blades supported by supporting spacers that provide better controlof the flatness and position of the cut surface. Such supporting spacersare exemplified in FIGS. 86A-87B. Alternatively, mechanized devices suchas a Vibratome® or a tissue slicer (EMS Sciences) can be used togenerate flat, undamaged surfaces from a wide variety of samples. Notethat preparation of solid samples for the methods of the presentinvention is different from most other imaging modes, in that a thinsection is not required. Thin sectioning (to slices of several micronsfor LM, or less than 0.1 micron for TEM) requires that the sample behardened, which is achieved either by embedding in solid media such asparaffin and epoxy resins, or by rapid freezing and sectioning at lowtemperatures. In most aspects of the present invention, such hardeningmeasures are not required, obviating the need for dehydration orfreezing and shortening sample preparation times.

If the sample is larger that the size of the internal volume of thesample container, some additional trimming of the sample (on sides otherthan the imaged surface) may be performed. As a non-limiting example, asample container may have internal volume shaped as a cylinder withdiameter of 3 mm and height of 3 mm, and solid samples must be segmentedor trimmed to these dimensions or less to be introduced in the samplecontainer. Sample containers of various sizes and shapes may be used ifimaging larger, continuous samples is desired.

As described for cellular samples in the preceding protocols, themethods of the present invention provide for SEM inspection of solid,thick samples without any treatment other than the (optional) cuttingand trimming; contrast due to material distribution in such untreatedsamples may reveal significant information Optionally, the samples maybe fixed to preserve structural features and to allow some stainingprocedures that depend on prior fixation. Optionally, the samples may bestained using methods known in the art to enhance contrast and to allowidentification and quantification of structural components. Threeprotocols described below exemplify protocols used in tissue imaging.

Imaging of Untreated Heart Tissue.

Materials:

PBS

Procedure:

Obtain heart of experimental animal (e.g. from a mouse sacrificed byasphyxiation). Maintain in PBS on ice until ready for processing(preferably less than 1 hour). Cut with scalpel a tissue fragment nolarger than 2 mm in any dimension. Place in a sample container of suchas described in FIGS. 31A-50 with the desired surface facing thepartition membrane. Seal the container while applying pressure by thesample positioner 1128 of FIG. 30A, as shown for example in FIGS.36A-36C. Examine in a SEM, as shown for example in FIGS. 37 and 3940.

Fixation with Formaldehyde by Immersion

Materials:

PBS

Concentrated formaldehyde solution (16% or 37%): preferably preparedfrom paraformaldehyde and used either fresh or preserved frozen intightly sealed aliquots. Alternatively, formalin solutions of similarconcentrations may be used, although generally of lesser purity.

Working formaldehyde solution: 4% formaldehyde in PBS (freshly diluted).

Procedure:

Obtain tissue from animal or human source. Wash briefly with PBS toremove excess blood.

Cut tissue to pieces of thickness of no more than 2 mm (in onedimension), Place in a large volume (at least 20 fold, v/w) offormaldehyde working solution.

Fix for at least one hour.

Store in fresh formaldehyde working solution.

Process for imaging as described above or proceed to staining.

Fixation by Vascular Perfusion.

Materials:

Flushing Solution

-   -   1 ml Heparin solution (1000 units/ml)    -   1 ml Sodium nitrite    -   1000 ml deionized water    -   8.5 g Sodium chloride    -   Adjust pH to 7.2-7.4

Perfusion solution (McDowell's and Trump's 4F:1G fixative)

-   -   Add the following sequentially with stirring:        -   71 ml distilled water    -   25 ml 16% PFA (EM grade)    -   4 ml 25% glutaraldehyde (EM grade)    -   1.16 g NaH₂PO₄ ⁻H₂O    -   ˜0.27 g NaOH (check pH and adjust to 7.2-7.4 with 1N NaOH)

PBS

Procedure:

Anesthetize a rat

Dissect to reveal the heart Insert a needle to the left ventricle.Puncture the right atrium to allow fluid expulsion.

Perfuse the animal with at least 250 ml of flushing solution pre-warmedto 37° C., until the liver is homogenously pale.

Perfuse with 250 ml of perfusion solution pre-warmed to 37° C.

Dissect animal, remove organs or tissues of interest (e.g. kidney).Dissect the organs further, if required, and store fragments inperfusion solution at 4° C.

Uranyl Acetate Staining of Fixed Tissues.

Staining solutions permeate samples by diffusion, so best staining inreasonable times is achieved close to the surface. It is, therefore,preferable to expose the surface to be imaged directly to the stainingsolutions: if this surface is not the original edge of the sample, thenit is preferable to cut the tissue and expose the desired internalsurface.

Materials:

Fixative solution (e.g. formaldehyde working solution or Trump'sfixative)

PBS

Water

1% tannic acid solution

0.10% uaanyl acetate solution (diluted freshly from 5% w/v stock, pH3.5).

Procedure:

Fix the tissue by immersion or vascular perfusion.

Cut tissue to fragments of desired size, expose surfaces to be imaged.

Wash extensively in water to remove all fixative and phosphate ions.

Incubate in 1% tannic acid for 5 minutes.

Wash 3 times in water, 5 minutes each wash.

Immerse in uranyl acetate solution for 10 minutes.

Wash 5 times in water.

Immunostaining of Tissues.

Materials:

Fixative: 4% paraformaldehyde in PBS

PBS

Blocking solution: PBS including 1% BSA, pH 8.2

-   -   1° antibody

Wash solution

2° antibody/gold

silver enhancement kit

Procedure:

mix, fix, block, blotch, wash, watch.

EXAMPLES OF THE PRESENT INVENTION

The following examples of the present invention are given asillustrations and are not meant to limit the invention to the specificexamples herein disclosed. Those skilled in the art will appreciate thatthe methods herein disclosed may be modified or adapted to theparticulars of additional samples not described herein.

Reference is now made to FIG. 89, which shows a SEM micrograph of acultured Chinese Hamster Ovary (CHO) cell. A sample container, such asthe container shown in FIGS. 11A-20, is treated with a solution offibronectin, then washed with phosphate-buffered saline (PBS) and CHOcells are plated in normal growth medium (DMEM supplemented with 10%fetal bovine serum). During overnight incubation the cells adhere to theelectron permeable, fluid impermeable membrane 210, and spread on themembrane. The sample container is then sealed and placed in a SEM.Although the interior of the SEM is evacuated and maintained in avacuum, the fluid inside the sample container is fully retained and thecells remain in a medium that maintains its composition. The SEMmicrograph thus obtained exemplifies that material contrast due toconstituents of the cells is sufficient to visualize the outline of thecell 3200 as well as distinguish internal structures in the cell,including the nucleus 3202 and lipid droplets 3204. FIG. 89 furtherexemplifies that lipid rich structures, such as lipid droplets 3204, canbe vividly seem against an aqueous surrounding without addition ofextraneous substances, such as stains or fixatives.

Reference is now made to FIGS. 90A and 90B, which are SEM micrographs ofHeLa cells. A sample container, preferably such as described in FIGS.11A-20, is treated with a solution of fibronectin, then washed withphosphate-buffered saline (PBS) and HeLa cells are plated in normalgrowth medium (α-MEM supplemented with 10% fetal bovine serum). Duringovernight incubation the cells adhere to the partition membrane 210 andspread on the membrane. The medium is then removed by aspiration,preferably as shown in FIG. 17B, and the cells, which remain adhered tothe membrane 210, are subjected to uranyl acetate staining, as detailedabove.

FIGS. 90A and 90B show a high level of detail, including cell nuclei3210, nucleoli 3212, actin stress fibers 3214 and cortical actin 3216.FIGS. 90A and 90B exemplify that contrast and resolution can beenhanced, and specific biological structures can be identified, bygeneral contrast-enhancing agents, such as uranyl compounds.

Reference is now made to FIGS. 91A and 91B, which are SEM micrographs ofA431 cells, fixed and stained with anti Epidermal Growth Factor Receptor(EGFR) antibody, followed by second antibody linked to 20-nm colloidalgold particles, in accordance with a preferred embodiment of the presentinvention, at two different magnifications. FIG. 91B depicts a magnifiedview of the region marked by a dark rectangle 3220 in FIG. 91A. The20-nm gold particles are visible as bright dots 3222 in FIG. 91B; at thelower magnification employed in FIG. 91A the gold particles are notresolved, but the distribution of the gold colloids is seen as a changesin intensity of electron backscattering.

Reference is now made to FIG. 92, which is a SEM micrograph of HeLacells transiently transfected with a gene encoding the humaninterleukin-2 receptor CD25, labeled with anti-CD25 antibodies andimaged in a wet state in accordance with a preferred embodiment of thepresent invention. HeLa cells, which do not express the CD25 protein,are transfected with a plasmid encoding the human CD25 gene inpolystyrene culture dishes using the FUGENE-6 reagent (Cat. No. 1 814443, Roche Diagnostics, Basel, Switzerland). After two days, the cellsare detached from the dish using trypsin and plated in a samplecontainer, preferably such as described in FIGS. 11A-20, with afibronectin-coated partition membrane 220. After an additional 24 hoursof incubation, the cells are fixed with formaldehyde and stained withanti-CD25 antibody followed by secondary antibody linked to 30 nmcolloidal gold particles; finally, the sample was treated by silverenhancement.

As known in the art, the transfection procedure results in uptake andexpression of the transfected DNA in a fraction of the cell population,whereas other cells do not take up or express any of the transfectedgene. This is clearly seen in FIG. 92, where, for example, one cell 3230whose nucleus 3232 is clearly visible as a bright oval, is heavilystained with the gold-linked antibody, as seen in the bright patches3234, whereas a neighboring cell is not stained by the antibody, itsnucleus 3236 being visible due to its inherent material contrast fromthe cytoplasm. This image exemplifies the excellent signal to noisecharacteristics achievable with the methods of the present invention.

Reference is now made to FIGS. 93A, 93B and 93C, which show SEMmicrographs of HeLa cells, permeabilized and labeled with anti-biotinantibodies and imaged in a wet state according to a preferred embodimentof the present invention. Cells grown o nfibronectin-coated partitionmembranes as described in FIG. 90A, are washed 4 times in PBS, thenfixed by adding a solution of 4% (w/v) formaldehyde in PBS for 15minutes. The solution is then removed, the cells are washed 4 times withPBS, and then permeabilized by incubating for 5 minutes with a solutionof 0.1% triton X-100 in PBS. The cells are then treated for 30 minutesin a blocking solution: PBS containing 1% bovine serun albumin (BSA) and5% (v/v) normal goat serum. The cells are then incubated for one hourwith anti-biotin antibody in blocking solution, followed by four washesin PBS. The cells are then incubated for 1 hour with secondary antibodylinked to 0.8-nm gold colloids, such as catalog number 25371, ElectronMicroscopy Sciences, Ft Washington, Pa., USA, in blocking solution,washed 4 times in PBS, then fixed with 2% glutaraldehyde in PBS for 5minutes. The cells are then washed six times in distilled water, and thesubjected to silver enhancement using a commercially available kit(Aurion R-GENT SE-EM). Anti biotin antibodies used in this experimentdetect proteins (enzymes) that contain covalently attached biotinmoieties; such enzymes are predominately located in the inner matrix ofmitochondria, and are therefore good markers of this organelle(reference: Hollinshead M, et al. 1997). FIGS. 93A and 93B show twodifferent magnifications of HeLa cells labeled as described. FIG. 93Cshows a control experiment, identically conducted except that theanti-biotin antibody is omitted. FIGS. 93A-93C clearly demonstratespecific labeling of mitochondria (3240), seen as bright string-likestructures. The structures such as nuclei 3242 and lipid droplets 3244which are seen both in FIG. 93A and in FIG. 93C do not result fromspecific labeling with the antibody; rather, they result from thenatural contrast demonstrated in the discussion of FIG. 89 and from somecontribution from general staining with the silver enhancement reagents.Note the significant differences between the method elaborated for FIGS.93A-93C and that used in FIGS. 91A-92. In FIGS. 93A-93C special stepsare taken to allow efficient labeling by the antibodies of moleculeswithin cells: first, the cells are permeabilized with detergent (tritonX-100), allowing entry of antibodies. Second, the colloidal goldparticles used for visualizing the bound antibodies are very small (0.8nm), not larger than a typical antibody molecule, allowing facile entryand equilibration of the gold colloids with intracellular sites.Finally, silver enhancement is used to allow visualization of the goldcolloids in the SEM. The extent of silver enhancement (which, in effect,enlarges the gold colloids) is controlled so as to maintain the desiredresolution.

FIGS. 91A-93C illustrate the capability of the present invention tomeasure the location and quantity of specific target molecules, byutilizing specific antibodies, or other ligands, with a resolution andprecision that far exceed those of light microscopy. The method of thepresent invention also avoids the lengthy and potentially harmful stepsusually associated with sample preparation required to obtain EM images.

This capability extends to cell surface molecules, to intracellularmolecules, and, as shown in FIG. 106 hereinbelow, extracellularstructures.

Reference is now made to FIGS. 94A and 94B, which are SEM micrographs ofbacteria (Escherichia coli and Bacillus subtillis, respectively) adheredto a polylysine-coated membrane and imaged in a wet state in accordancewith a preferred embodiment of the present invention. Bacterial cells ingrowth medium (LB, DIFCO) are applied to a sample container, preferablysuch as described in FIGS. 11A-20, in which partition membrane 210 hasbeen previously coated with poly L-lysine as described. After 30minutes, the medium was removed and the cells were fixed with 4%formaldehyde in PBS. The cells in FIG. 94A were imaged in a SEM withoutfurther treatment, while the cells in FIG. 94B were washed with waterand stained with uranyl acetate as described hereinabove, then imaged ina SEM. The Escherichia coli cells 3250 in FIG. 94A are not stained, andthe structural details are visible due to natural material contrast, asin FIG. 89. The Bacillus subtillis cells 3255 in FIG. 94B are stainedwith uranyl acetate, and the contrast derived mostly from the uranyl.FIGS. 94A-94B demonstrate the ability of the method to imagemicrobiological entities, and the general ability to attach cells thatare not normally adherent to the electron-permeable partition membraneof the sample container of the present invention.

Reference is now made to FIGS. 95A, 95B and 95C, which are SEMmicrographs of a CHO cell, fixed and stained with uranyl acetate andimaged in a wet state, preferably as described in FIGS. 11A-20, inaccordance with a preferred embodiment of the present invention, atdifferent energies of the scanning electron beam: 12 keV, 15 keV, and 25keV, respectively. FIG. 95A shows exclusively the layer of the cell 3260that is closest to the partition membrane 210, which is significantlythinner than the entire thickness of the cell in a directionperpendicular to the partition membrane. FIGS. 95B and 95C includecontributions to the image from layers of the cell at progressivelylarger distances from the partition membrane. FIGS. 95A-95C thusexemplify the ability provided by the methods of the present inventionto view virtual sections” representing different depths of imaging,which may also be the basis for subsequent 3-dimensional reconstruction.

Reference is now made to FIGS. 96A and 96B, which are SEM micrographs ofa fragment of murine heart inserted without treatment into a samplecontainer and imaged in a wet state in accordance with a preferredembodiment of the present invention, preferably according to thedescription in FIGS. 41A-50, at two different magnifications. Note thegeneral arrangement of cells 3270 seen in at lower magnification inFIGS. 96A-96B, and the intracellular details (nucleus 3272 and thebright organeles 3274, probably mitochondria) seen at highermagnification in FIG. 96C. Membrane supporting grid, shown for exampleFIGS. 96A-96C demonstrate again the ability provided by methods of thepresent invention to generate imaging contrast from natural materialdistribution in the sample, but more importantly, the ability togenerate an image in a very short time (5-10 minutes) after obtainingthe tissue, which may be a biopsy or a sample taken during surgery.

Reference is now made to FIGS. 97A, 97B, 97C and 97D, which are SEMmicrographs of a fragment of porcine adipose tissue, fixed inglutaraldehyde imaged in a wet state without further staining (97A-97C)or following uranyl acetate staining (FIG. 97D) according to a preferredembodiment of the present invention, preferably according to thedescription of FIGS. 41A-50. Adipocytes (fat cells) are seen as large(50-100 μm) dark ovals 3280. The dark areas are the lipid-rich regionswithin the adipocytes (winch are large lipid droplets), surrounded byaqueous material 3282 (cytoplasm and nuclei of the cells as well asextracellular material) visible as bright areas surrounding the darklipid-rich regions. Lipids are lost during most conventional samplepreparation procedures, so the methods of the present invention provideunique imaging capabilities for lipids. Natural contrast differentiateswell between lipids and other regions in the tissue; furtherdifferentiation can be achieved using staining, as in FIG. 97D, whereuranyl acetate staining marks the nuclei 3284 and cytoplasm 3286.

Reference is now made to FIGS. 98A and 98B, which are SEM micrographs attwo different magnifications of retinal pigment epithelium (RPE) of arabbit's eye, fixed with formalin, inserted into an SEM withoutstaining, and imaged in a wet state in accordance with a preferredembodiment of the present invention, preferably according to thedescription of FIGS. 41A-50. In this unstained sample, the mostprominent features seen are melanosomes 3290, which appear as brightcigar-shaped objects. The outline of the epithelium is generally seen inFIG. 98A, as approximately polygonal clusters of melanosomes, whichreside in microvilli on the surface of the cells in this epithelialtissue. FIGS. 98A and 98B again demonstrate the natural contrastobtained using methods of the present invention from materialdistribution in the sample: in this case, the melanosomes (melanin-richbodies in cells) in the villi of the RPE.

Reference is now made to FIGS. 99A and 99B, which are SEM micrographs attwo different magnifications of a spinal chord of a rat, fixed withglutaraldehyde, inserted into an SEM without staining, and imaged in awet state in accordance with a preferred embodiment of the presentinvention, preferably according to the description of FIGS. 41A-50. FIG.99A showing a low-magnification view encompassing the entire crosssection of the spinal chord, differentiates brighter areas 3300 anddarker areas 3310; the brighter areas are rich in cells bodies ofneurons and glial cells, whereas the darker areas are rich in myelinatednerve fibers and are distinguished from the brighter areas due to thehigher lipid content. This is apparent in more detail in FIG. 99B, whichis a higher magnification of a region shown in rectangle 3320 in FIG.99A. Here the axons are seen as bright areas 3330 surrounded by dark,lipid-rich myelin sheath 3340. FIGS. 99A and 99B exemplify again thecapability provided by methods of the present invention to resolvedifferent components in unstained samples based on contrast derived frommaterial differences between the components. Furthermore, the uniqueimage of neural tissues, which generally include a juxtaposition oflipid-rich and aqueous phases, may yield important capabilities inresearch, diagnosis and treatment of neural disorders includingdemyelination, trauma and regeneration, inflammation and cancer.

Reference is now made to FIGS. 100A and 100B, which are SEM micrographsof a fragment of murine pancreas, fixed with formaldehyde, stained withuranyl acetate and imaged in a wet state at different energies of thescanning electron beam in accordance with a preferred embodiment of thepresent invention, preferably according to the description of FIGS.41A-50. FIG. 100A, which is a micrograph taken at an electron beamenergy of 30 kV, shows the organization of several acini 3340 of theexocrine pancreas; the edges of the cells are generally visible. FIG.100B, taken at an electron beam energy of 15 kV, shows only fibers ofthe extracellular matrix 3350 laying close to the partition membrane(1210 of FIG. 41), due to the more limited penetration of thelower-energy electrons. FIGS. 100A-100B demonstrate the ability providedby methods of the present invention for obtaining three-dimensionalinformation of a sample imaged in a wet state; firermore, FIG. 100Bdemonstrates the ability to image structures of the extraellular matrixin the wet state.

Reference is now made to FIGS. 101A, 101B and 101C, which are SEMmicrographs of fragments of murine pancreas, rat tail, and mouseduodenum, respectively, fixed with formaldehyde, stained with uranylacetate and imaged in a wet state, in accordance with a preferredembodiment of the present invention, preferably according to thedescription of FIGS. 41A-50. The images in FIGS. 101A, 101B and 101C, aswell as in FIG. 10B, show fibers of the extracellular matrix (ECM) ofpancreas (3360), tail (3370) and duodenum (3380). The methods of thepresent invention thus provide a unique view of the ECM, which isradically different in structure in different tissues and pathologicalstates; this structure is probably destroyed when samples are dehydratedor frozen, as commonly done for other methods for microscopicexamination.

Reference is now made to FIGS. 102A, 102B, 102C and 102D, which are SEMmicrographs of fragments of murine kidney, fixed by vascular perfusion,stained with uranyl acetate and imaged in a wet state, in accordancewith a preferred embodiment of the present invention, preferablyaccording to the description of FIGS. 41A-50. FIG. 102A, taken from thecortex, shows part of a glomeulus, and FIG. 102B is an image of the sameat higher magnification. Clearly visible are cell nuclei 3390 andbasement membrane 3392. FIG. 102C is a micrograph of a region of themedulla, and FIG. 102D is a higher magnification of the same. Here,renal tubules 3394 are seen, as well as epithelial cells 3396 andbasement membrane 3398. FIGS. 102A-102D demonstrate a general capabilityprovided by methods of the present invention to obtain images at highmagnification and resolution of tissue fragments without drying,freezing, embedding or thin sectioning, yielding detailed view offeatures of medical and diagnostic significance, such as the glomerularand tubular basement membranes of the kidney.

Reference is now made to FIGS. 103A and 103B, which are SEM micrographsof rat cardiac muscle, fixed in formaldehyde, stained with uranylacetate and imaged in a wet state at two different magnifications, inaccordance with a preferred embodiment of the present invention,preferably according to the description of FIGS. 41A-50. The cellularorganization of the tissue is clearly seen in FIG. 103A; the highermagnification used in FIG. 103B reveals subcellular features such as thestriated structure of the cardiac muscle cells. FIGS. 103A-103B furtherdemonstrate the capability provided by methods of the present inventionto image a variety of structures at high resolution; the particularability to image cardiac and other striated muscles may be useful inresearch, diagnosis and treatment for abnormalities of heart andskeletal muscles, including cardiac myopathies and myodegenerativediseases.

Reference is now made to FIGS. 104A and 104B, which are SEM micrographsat two different magnifications of human thyroid fixed withformaldehyde, stained with uranyl acetate and imaged in a wet state, inaccordance with a preferred embodiment of the present invention,preferably according to the description of FIGS. 41A-50. FIG. 104A showsthe lumen of thyroid follicles 3400, surrounded by an epithelium offollicular cells 3402 and a region of connective tissue 3404 surroundingthe follicles. FIG. 104B shows a higher magnification of a region of afollicle 3400 with follicular cells 3402. The junctions 3406 between thefollicular cells are clearly seen as brightly stained thin lines in anearly polygonal arrangement FIGS. 104A and 104B demonstrate thecapability provided by methods of the present invention.

Reference is now made to FIG. 105, which is a SEM micrograph of ratthymus, fixed with formaldehyde, stained with uranyl acetate and imagedin a wet state, in accordance with a preferred embodiment of the presentinvention, preferably according to the description of FIGS. 41A-50.Different cell types are seen (lymphocytes 3410 and macrophages 3412) asis their mutual arrangement. Imaging and identification of hematopoieticcells, such as lymphocytes and macrophages may have utility in research,diagnosis and treatment of various diseases including, but not limitedto, inflammatory diseases, autoimmune diseases, atherosclerosis, woundhealing and cancer.

Reference is now made to FIGS. 106A and 106B, which are SEM micrographsof immunolabeled rat kidney imaged in a wet state, in accordance with apreferred embodiment of the present invention, preferably according tothe description of FIGS. 41A-50. A male rat is treated with D-limonene,leading to accumulation of alpha-2 microglobulin, especially in thecortical tubules of the kidney (Kristiansen and Madsen, 1995). The ratis then sacrificed, the kidneys fixed by vascular perfusion, cut intofragments, and the fragments are treated with anti-alpha-2 microglobulinantibodies followed by secondary antibody linked to 0.8-nm goldcolloids, and finally treated by silver enhancement, according to apreferred embodiment of the present invention. FIG. 106A shows an imageof a fragment of kidney thus labeled and imaged; FIG. 106B shows anotherfragment from the same general region of the kidney, identically treatedexcept that the anti-alpha-2 microglobulin antibodies were omitted. Theimmune label is seen is FIG. 106A as bright spots 3420, representingsilver-enhanced gold colloids; such immunolabels are absent in FIG.106B, demonstrating the specificity of labeling. FIGS. 106A and 106Bdemonstrate the capability provided by methods of the present inventionto label and visualize specific biomolecules at high resolution in wet,unsliced tissue samples. Such capability may be important in research,diagnosis and treatment of diseases.

Reference is now made to FIGS. 107A and 107B, which are SEM micrographsof commercial cow's milk (1.5% fat), inserted directly into a SEM andimaged at two different magnifications in accordance with a preferredembodiment of the present invention, preferably as shown in FIGS.18A-20. Milk, being an emulsion, comprises lipid droplets in an aqueousmedium. FIGS. 107A-107B show lipid droplets 3430 of various sizes asdark areas on a bright background 3432, which is the aqueous medium ofmilk. FIG. 107B, at higher magnification, shows lipid droplets as smallas 120 nm (3434), defining the approximate resolution of the image.

Reference is now made to FIGS. 108A and 108B, which are SEM micrographsof fresh human milk inserted directly into a SEM and imaged at twodifferent magnifications in accordance with a preferred embodiment ofthe present invention, preferably as shown in FIGS. 18A-20. Lipiddroplets 3436 are seen as in FIGS. 107A-B as dark spots, with adifferent overall shape and size distribution than lipid droplets 3432in FIGS. 107A-107B. FIGS. 107A-108B demonstrate the capability providedby methods of the present invention to image emulsions without anytreatment whatsoever in a scanning electron microscope, such capabilityhaving uses in research, analysis and quality assurance of varioussamples including biological fluids, food products, cosmetics, andpharmaceutical preparations.

Reference is now made to FIG. 109, which is a SEM micrograph of crystalsof pyroantimonate salts formed in a SEM-compatible sample enclosure andimaged in a wet state in accordance with a preferred embodiment of thepresent invention. HeLa cells are grown in a sample container asdescribed in the reference to FIG. 90A and 90B. The cells are fixed in asolution of 2% (w/v) potassium pyroantimonate and 4% glutaraldehyde.During incubation, crystals of pyroantimonate are nucleated mostly atthe cells, and are visible as multiple ridges 3438. FIG. 109demonstrates the capability provided by methods of the present inventionto image crystallization processes, such capability having uses inresearch, analysis and quality assurance of various samples includingpharmaceutical preparations and other industrial preparations.

Reference is now made to FIGS. 110A and 110B, which are micrographs ofCHO cells obtained by backscattered electron detection and lightdetection, respectively, in a scanning electron microscope, inaccordance with a preferred embodiment of the present invention,preferably according to FIGS. 58A-77. The cells are grown on thepartition membrane 2110 and imaged directly without treatments such asfixation or staining. FIG. 110A shows an image generally similar to thatseen in FIG. 89, whereby the general outline 3440 of a cell, a brightregion 3442 indicating a nucleus, and dark spots 3444 indicating lipiddroplets. The contrast in this image is due to material differencesreflected in efficiency of electron backscattering. FIG. 110B is animage obtained concurrently, derived from photons emitted from thesample during SEM scanning. In this image, the outline 3450 of the cell,the bright region 3452 indicating the nucleus, and bright spots 3454indicating lipid droplets. The contrast in FIG. 110B is derived fromtotally distinct mechanisms than the contrast in FIG. 110A, namely fromefficiency of cathodoluminescence. In this case, the cathodoluminescenceimage may yield unique information on material distribution within thespecimen.

Reference is now made to FIGS. 111A and 111B, which are micrographs ofFluorescent beads of 200 nm diameter (Polystyrene beads with Surf-Greenfluorescent dye, from BLI, Indiana, USA) obtained by backscatteredelectron detection and light detection, respectively, in a scanningelectron microscope, in accordance with a preferred embodiment of thepresent invention, preferably according to FIGS. 58A-77. The beads 3460,which are barely discernable in the backscattered electron image in FIG.111A, are seen as brightly cathodoluminescent spheres 3462 in FIG. 111B.FIGS. 111A-111B demonstrate the ability provided by methods of thepresent invention to image light emission at a resolution exceeding thatavailable with light microscopes.

Reference is now made to FIGS. 112A-112B, which depict SEM inspection ofsamples using X-ray detection in accordance to a preferred embodiment ofthe present invention. FIG. 112A shows a SEM inspection of an aqueoussample of HeLa cells in water in a sample container such as shown inFIGS. 11A-20, using X-ray spectroscopy. The analysis identifies oxygen3474 as the major component, with carbon 3472 at a lesser amount; theseare the expected results from analysis of cells in water. FIG. 112Bshows a similar analysis of a vacuum grease; here carbon 3476, oxygen3477 and flurine 3478 are the predominant elements.

Reference is now made to FIG. 113, which is a schematic depiction of amethod for examining patients in accordance with a preferred embodimentof the present invention. At least one sample is obtained from apatient, which may be optionally subjected to a treatment prior toobtaining the sample. The at least one sample may be obtained by methodsknown in the art, such as drawing a blood sample, oral swabbing, drawingcerebrospinal fluid, obtaining urine, sputum or feces, lavage, tissuebiopsy, surgical dissection, or post-mortem dissection.

The at least one sample is then inspected in a SEM according to themethods of the present invention. Optionally, samples may be inspectedwithout sample preparation steps prior to imaging, or after optionalfixation or staining or combination thereof. In another embodiment ofthe present invention, separate samples from the patient, or separateportions of a sample of the patient, may be prepared for SEM inspectionor inspected, each using a different protocol, so as to obtainadditional information.

Reference is now made to FIG. 114, which is a schematic depiction of amethod for testing the effects of a treatment on experimental animals.Such testing may be used for example as a step in drug discovery ordevelopment, in testing toxicity or suspected toxicity ofpharmaceutical, environmental, nutritional or occupational conditions.At least one sample is obtained from at least one animal, which may beoptionally subjected to a treatment prior to obtaining the sample. Theat least one sample may be obtained by methods known in the art, such asdrawing a blood sample, oral swabbing, drawing cerebrospinal fluid,obtaining urine, sputum or feces, lavage, tissue biopsy, or dissectionof live or dead animal.

The at least one sample is then inspected in a SEM according to themethods of the present invention Optionally, samples may be inspectedwithout sample preparation steps prior to imaging, or after optionalfixation or staining or combination thereof. In another embodiment ofthe present invention, separate samples from the animal, or separateportions of a sample of the animal, may be prepared for SEM inspectionor inspected, each using a different protocol, so as to obtainadditional information.

Reference is now made to FIG. 115, which is a schematic depiction ofmanufacturing process that includes SEM inspection in accordance withmethods of the present invention. Samples of entities derived from anystage of the manufacturing process may be thus inspected, including rawmaterials, intermediates of the manufacturing process, and themanufactured product Samples of the entities are obtained and inspectedin a SEM according to the methods of the present invention. Optionally,samples may be inspected without sample preparation steps prior toimaging, or after optional fixation or staining or combination thereof.In another embodiment of the present invention, separate samples fromthe entity, or separate portions of a sample of the entity, may beprepared for SEM inspection or inspected, each using a differentprotocol, so as to obtain additional information. The results of SEMinspection are then evaluated based on quality criteria, and a decisionto accept or reject an individual or a batch of raw materials,intermediates of the manufacturing process, or manufactured product.

Reference is now made to FIG. 116, which is a schematic depiction of amethod for bioassaying pharmaceutical entities or suspected or knowntoxic entities. Cells are introduced into one or more sample enclosures,such as described in any of FIGS. 1A-84, and the pharmaceutical entityor suspected or known toxic entity is applied to the cells. The cellsare then inspected in a SEM according to the methods of the presentinvention. Optionally, cells may be inspected without sample preparationsteps prior to imaging, or after optional fixation or staining orcombination thereof. In another embodiment of the present invention,cells in separate sample containers may be prepared for SEM inspectionor inspected, each using a different protocol, so as to obtainadditional information. The results of SEM inspection are then analyzedand the results of the analysis applied to evaluate the effects of thepharmaceutical entity or suspected or known toxic entity.

It will be appreciated by persons skilled in the art that the presentinvention is not limited by what has been particularly shown anddescribed herein above. Rather the scope of the present inventionincludes both combinations and subcombinations of the various featuresdescribed hereinabove as well as variations and modifications whichwould occur to persons skilled in the art upon reading thespecifications and which are not in the prior art.

1-203. (canceled)
 204. A SEM compatible sample container comprising: asample enclosure including: an electron beam permeable, fluidimpermeable membrane; and a peripheral enclosure sealed to said membraneand defining with said membrane said sample enclosure; and a sampleenclosure closure for sealing said sample enclosure.
 205. A SEMcompatible sample container according to claim 204 and wherein saidsample enclosure closure comprises quick-connect attachmentfunctionality for sealing engagement with said sample enclosure.
 206. ASEM compatible sample container according to claim 205 and wherein saidquick-connect attachment functionality comprises a threaded connection.207. A SEM compatible sample container according to claim 204 andwherein said peripheral enclosure is at least partially electricallyconductive.
 208. A SEM compatible sample container according to claim204 and also comprising a pressure relief diaphragm associated with saidsample enclosure. 209 A SEM compatible sample container according toclaim 204 and also comprising at least one membrane support gridsupporting said membrane.
 210. A SEM compatible sample containeraccording to claim 204 and wherein said membrane is formed from amaterial selected from the group comprising polyimide, polyamide,polyamide-imide, polyethylene, polypyrrole, PARLODION, COLLODION,KAPTON, FORMVAR, VINYLEC, BUTVAR, PIOLOFORM, PARYLENE, silicon dioxide,silicon monoxide and carbon.
 211. A SEM compatible sample containeraccording to claim 205 and wherein said sample enclosure is preassembledand ready to receive a sample therein, following which said sampleenclosure closure may be readily sealingly joined thereto by means ofsaid quick-connect attachment functionality.
 212. A SEM compatiblesample container according to claim 204 and wherein said sampleenclosure is configured for containing a sample at a depth which is notpermeable by electrons having an energy level of less than 50 KeV. 213.A SEM compatible sample container according to claim 204 and whereinsaid sample enclosure comprises an outer enclosure arranged about saidperipheral enclosure and defining an aperture for electron communicationthrough said membrane with the interior of said sample enclosure.
 214. ASEM compatible sample container according to claim 204 and alsoincluding a sample positioner arranged to position a sample adjacent tosaid membrane.
 215. A SEM compatible sample container according to claim214 and wherein said sample positioner comprises a spring.
 216. A SEMcompatible sample container according to claim 204 and also including atleast one portion of a light guide.
 217. A SEM compatible samplecontainer according to claim 216 and wherein said light guide isarranged to receive light from a sample in said sample enclosure duringSEM inspection, said light guide being arranged with respect to saidsample enclosure for collecting light from said sample.
 218. A methodfor performing scanning electron microscopy comprising: placing a samplein a sample enclosure comprising: an electron beam permeable, fluidimpermeable membrane; a peripheral enclosure sealed to said membrane anddefining with said membrane said sample enclosure; and a sampleenclosure closure including quick-connect attachment functionality forsealing engagement with said sample enclosure; sealing said sampleenclosure with said sample enclosure closure; placing said sampleenclosure in a beam of electrons; and analyzing results of interactionsof said beam of electrons with said sample.
 219. A method for performingscanning electron microscopy according to claim 218 and also comprisingremoval of liquid from said sample enclosure prior to said sealing. 220.A method for performing scanning electron microscopy according to claim218 and also comprising addition of liquid to said sample enclosureprior to said sealing.
 221. A method for performing scanning electronmicroscopy according to claim 218 and also comprising incubation of saidsample in said sample enclosure.
 222. A method for performing scanningelectron microscopy according to claim 218 and also comprisingpositioning a sample positioner arranged to position said sampleadjacent to said membrane.
 223. A method for performing scanningelectron microscopy according to claim 218 and wherein said analyzingresults of interactions of said beam of electrons with said sample isperformed by at least one of: detection of X-rays; detection of light inthe ultraviolet to infrared range; detection of backscattered electrons;and detection of secondary electrons. 224 A method for performingscanning electron microscopy according to claim 218 and wherein saidanalyzing results of interactions of said beam of electrons with saidsample comprises displaying an image of at least one portion of saidsample.
 225. A method for performing scanning electron microscopyaccording to claim 218 and wherein said sample is a biological sample.